| Tissue hypoxia and ischemia initiate events that lead to pre-retinal angiogenesis. Adenosine modulates a variety of cellular functions by interacting with specific cell surface G-protein coupled receptors (A 1, A2A, A2B, A3) and is a potential mediator of angiogenesis. The A2B receptor has been implicated in the mediation of angiogenesis. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design and characterize a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Active and inactive ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. HEK 293 cells were transfected with these plasmids, and A2B mRNA levels were determined by quantitative RT-PCR. Human retinal endothelial cells (HREC) were also transfected, and cell migration was examined. The effects of these ribozymes on the levels of pre-retinal neovascularization were determined using a mouse model of oxygen-induced retinopathy. We produced a ribozyme with a Vmax of 10.8 pmole min−1 and a kcat of 36.1 min 1. Transfection of HEK 293 cells with the plasmid expressing ribozyme resulted in a reduction of A2B mRNA levels by 45%. Transfection of HREC reduced NECA stimulated migration of the cells by 47%. Intraocular injection of the constructs into the mouse model reduced pre-retinal neovascularization by 54%. Our results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor, and provide further support for the role of the A2B receptor in retinal angiogenesis. |
