Proliferation, migration, and ERK activation in human retinal endothelial cells through A(2B) adenosine receptor stimulation

PURPOSE. The nucleoside adenosine has been implicated in angiogenesis. A pr evious study demonstrated that activation of the A(2B) adenosine receptor ( AdoR) increases cAMP accumulation, cell proliferation, and VEGF expression in human retinal endothelial cells (HRECs). In the present study, the role of this receptor was further characterized by examination of the effects of the selective A(2B) AdoR antagonists 3-N-propylxanthine (enprofylline) and 3-isobutyl-8-pyrrolidinoxanthine (IPDX) on AdoR-mediated HREC proliferatio n, capillary tube formation, and signal-transduction pathways. METHODS. HRECs were exposed to the adenosine analogue 5 ' -N-ethylcarboxami do-adenosine (NECA) in the absence or presence of AdoR antagonists. Migrati on was measured using Boyden chambers. Proliferation was assessed by counti ng cells. Western analysis was used to assess extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) in cell lysat es. The effect of AdoR activation on tube formation was studied using cells grown on a synthetic basement membrane matrix. RESULTS. NECA induced proliferation in a concentration-dependent manner tha t was inhibited by enprofylline and IPDX. NECA stimulated chemotaxis in a c oncentration-dependent manner that was also blocked by both A(2B) AdoR anta gonists. NECA activated ERK and CREB in HRECs. Both A(2B) AdoR antagonists diminished activation of ERK by NECA exposure. ERK activation was also bloc ked by the ERK-mitogen-activated protein kinase (MAPK) inhibitor PD98059, b ut not by the protein kinase A (PKA) inhibitor H-89. CREB activation was bl ocked by H-89, but not by PD98059, suggesting that ERK activation is indepe ndent of PKA. NECA enhanced tube formation on the matrix, whereas both A(2B ) AdoR antagonists attenuated this effect. CONCLUSIONS. The selective A(2B) AdoR antagonists, enprofylline and IPDX, i nhibited NECA-stimulated proliferation, ERK activation, cell migration, and capillary tube formation. A(2B) AdoR inhibition may offer a way to inhibit retinal angiogenesis and provide a novel therapeutic approach to treatment of diseases associated with aberrant neovascularization, such as diabetic retinopathy and retinopathy of prematurity.