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Abstract
PURPOSE: To determine the mechanistic role for adenosine and adenosine receptors in the hypoxic induction of vascular endothelial growth factor (VEGF) in retinal microvascular cells. METHODS: Bovine retinal capillary endothelial cells and microvascular pericytes were studied under normoxic (95% air, 5% CO2) or hypoxic conditions (0% to 2% O2, 5% CO2, 93% to 95% N2) using a variety of well-characterized adenosine and adenosine receptor agonists and antagonists. Vascular endothelial growth factor mRNA expression was evaluated by Northern blot analysis, VEGF protein levels were determined by Western blot analysis, and cyclic adenosine monophosphate (cAMP) accumulation was measured by radioimmunoassay. RESULTS: Inhibitors of oxidative respiration increased VEGF mRNA 5 +/- 3 times (P < 0.001) after 3 hours. Adenosine A1 receptor (A1R) agonist N6-cyclopentyl-adenosine did not increase VEGF mRNA at A1R stimulatory concentrations; however, adenosine A2 receptor (A2R) agonists DPMA, NECA, and CGS21680 increased VEGF mRNA in a dose-dependent manner with elevations of 2 +/- 0.3 (P < 0.001), 2.3 +/- 0.5 (P = 0.016), and 2 +/- 0.2 (P = 0.002) times, respectively. A2R antagonist CSC and adenosine degradation by adenosine deaminase reduced hypoxic stimulation of VEGF mRNA 68% +/- 18% (P = 0.038) and 37% +/- 6% (P = 0.025), respectively, in a dose-dependent manner. A1R antagonists DPCPX and 8-PT had no significant effect. Hypoxia and NECA increased VEGF protein secretion 4.7 times, whereas CSC inhibited hypoxia-induced VEGF protein secretion by 96%. NECA and CGS21680 increased cAMP production within 10 minutes, and cAMP stimulation increased VEGF mRNA 4.8 +/- 2.6 times (P = 0.034). CSC suppressed the hypoxic elevation of cAMP (P < 0.05). Inhibition of protein kinase A using H-89 reduced hypoxia-induced VEGF expression 61% +/- 6.3% (P = 0.043) in a dose-dependent manner. CONCLUSIONS: These data suggest that the hypoxia-induced accumulation of adenosine stimulates VEGF gene expression through stimulation of adenosine A2a receptor and subsequent activation of the cAMP-dependent protein kinase A pathway in retinal vascular cells.