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(Circulation Research. 2003;93:500.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Reduction in Preretinal Neovascularization by Ribozymes That Cleave the A_2B Adenosine Receptor mRNA

A. Afzal, L.C. Shaw, S. Caballero, P.E. Spoerri, A.S. Lewin, D. Zeng, L. Belardinelli, M.B. Grant

From the Departments of Pharmacology and Therapeutics (A.A., L.C.S., S.C., P.E.S., M.B.G.), and Molecular Genetics and Microbiology (A.S.L.), University of Florida, Gainesville, Fla; CVTherapeutics, Inc (D.Z., L.B.), Palo Alto, Calif.

Correspondence to M.B. Grant, MD, Professor, Department of Pharmacology and Therapeutics, University of Florida, PO Box 100267, Gainesville, FL 32610-0267. E-mail grantma{at}pharmacology.ufl.edu

Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein–coupled receptors (A₁, A_2A, A_2B, and A₃) and is a potential mediator of angiogenesis through the A_2B receptor. The lack of a potent, selective A_2B receptor inhibitor has hampered its characterization. Our goal was to design a hammerhead ribozyme that would specifically cleave the A_2B receptor mRNA and examine its effect on retinal angiogenesis. Ribozymes specific for the mouse and human A_2B receptor mRNAs were designed and cloned in expression plasmids. Human embryonic kidney (HEK) 293 cells were transfected with these plasmids and A_2B receptor mRNA levels were determined by quantitative real-time RT-PCR. Human retinal endothelial cells (HRECs) were also transfected and cell migration was examined. The effects of these ribozymes on the levels of preretinal neovascularization were determined using a neonatal mouse model of oxygen-induced retinopathy (OIR). We produced a ribozyme with a V_max of 515±125 pmol/min and a K_cat of 36.1±8.3 min^-1 (P1x10^-5). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A_2B receptor mRNA levels by 45±4.8% (P=5.1x10^-5). Transfection of HRECs reduced NECA-stimulated migration of cells by 47.3±1.2% (P=7x10^-4). Intraocular injection of the constructs into the mouse model reduced preretinal neovascularization by 53.5±8.2% (P=4.5x10^-5). Our results suggest that the A_2B receptor ribozyme will provide a tool for the selective inhibition of this receptor and provide further support for the role of A_2B receptor in retinal angiogenesis.

Key Words: adenosine receptors • neovascularization • ribozymes • retinopathy

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