Afzal A, Shaw L C, Caballero S, Spoerri P E, Lewin A S, Zeng D, Belardinelli L, Grant M B
Department of Pharmacology and Therapeutics, University of Florida, PO Box 100267, Gainesville, FL 32610-0267, USA.
Circ Res. 2003 Sep 19;93(6):500-6. doi: 10.1161/01.RES.0000091260.78959.BC. Epub 2003 Aug 14.
Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein-coupled receptors (A1, A2A, A2B, and A3) and is a potential mediator of angiogenesis through the A2B receptor. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. Human embryonic kidney (HEK) 293 cells were transfected with these plasmids and A2B 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 Vmax of 515+/-125 pmol/min and a Kcat of 36.1+/-8.3 min(-1) (P< or =1x10(-5)). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A2B 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 A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor and provide further support for the role of A2B receptor in retinal angiogenesis.
腺苷通过与特定的细胞表面G蛋白偶联受体（A1、A2A、A2B和A3）相互作用来调节多种细胞功能，并且是通过A2B受体介导血管生成的潜在介质。缺乏有效的、选择性的A2B受体抑制剂阻碍了对其特性的研究。我们的目标是设计一种锤头状核酶，它能特异性切割A2B受体mRNA，并研究其对视网膜血管生成的影响。设计了针对小鼠和人类A2B受体mRNA的核酶，并将其克隆到表达质粒中。用这些质粒转染人胚肾（HEK）293细胞，并通过定量实时逆转录聚合酶链反应（RT-PCR）测定A2B受体mRNA水平。也转染人视网膜内皮细胞（HREC）并检测细胞迁移。使用氧诱导视网膜病变（OIR）的新生小鼠模型确定这些核酶对视网膜前新生血管水平的影响。我们制备了一种核酶，其Vmax为515±125 pmol/分钟，Kcat为36.1±8.3分钟-1（P≤1×10-5）。用表达核酶的质粒转染HEK293细胞可使A2B受体mRNA水平降低45±4.8%（P=5.1×10-5）。转染HREC可使NECA刺激的细胞迁移减少47.3±1.2%（P=7×10-4）。将构建体眼内注射到小鼠模型中可使视网膜前新生血管减少53.5±8.2%（P=4.5×10-5）。我们的结果表明，A2B受体核酶将为选择性抑制该受体提供一种工具，并为A2B受体在视网膜血管生成中的作用提供进一步支持。
