Proliferation, migration, and ERK activation in human retinal endothelial cells through A(2B) adenosine receptor stimulation.
OBJECTIVE
The nucleoside adenosine has been implicated in angiogenesis. A previous 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 proliferation, capillary tube formation, and signal-transduction pathways.


METHODS
HRECs were exposed to the adenosine analogue 5'-N-ethylcarboxamido-adenosine (NECA) in the absence or presence of AdoR antagonists. Migration was measured using Boyden chambers. Proliferation was assessed by counting cells. Western analysis was used to assess extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) in cell lysates. 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 that was inhibited by enprofylline and IPDX. NECA stimulated chemotaxis in a concentration-dependent manner that was also blocked by both A(2B) AdoR antagonists. NECA activated ERK and CREB in HRECs. Both A(2B) AdoR antagonists diminished activation of ERK by NECA exposure. ERK activation was also blocked by the ERK-mitogen-activated protein kinase (MAPK) inhibitor PD98059, but not by the protein kinase A (PKA) inhibitor H-89. CREB activation was blocked by H-89, but not by PD98059, suggesting that ERK activation is independent 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, inhibited 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.
Grant
M B
MB
Department of Medicine, University of Florida, Gainesville 32610-0267, USA. grantma@pharmacology.ufl.edu
Davis
M I
MI
Caballero
S
S
Feoktistov
I
I
Biaggioni
I
I
Belardinelli
L
L
D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D015153:Blotting, Western; D002196:Capillaries; D002452:Cell Count; D002455:Cell Division / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D058915:Purinergic P1 Receptor Antagonists; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D012171:Retinal Vessels / Q000378:metabolism*; D015398:Signal Transduction; D014665:Vasodilator Agents / Q000494:pharmacology
D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D058915:Purinergic P1 Receptor Antagonists; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1; D014665:Vasodilator Agents; D019830:Adenosine-5'-(N-ethylcarboxamide); D020928:Mitogen-Activated Protein Kinases