Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA

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

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein-coupled receptors (A1, A 2A, 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≤1×10 -5). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A2B receptor mRNA levels by 45±4.8% (P=5.1×10-5). Transfection of HRECs reduced NECA-stimulated migration of cells by 47.3±1.2% (P=7×10-4). Intraocular injection of the constructs into the mouse model reduced preretinal neovascularization by 53.5±8.2% (P=4.5×10-5). 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.

Original languageEnglish (US)
Pages (from-to)500-506
Number of pages7
JournalCirculation Research
Volume93
Issue number6
DOIs
StatePublished - Sep 19 2003
Externally publishedYes

Fingerprint

Adenosine A2B Receptors
Catalytic RNA
Messenger RNA
Plasmids
Cell Movement
Transfection
Endothelial Cells
Adenosine-5'-(N-ethylcarboxamide)
Intraocular Injections
HEK293 Cells
G-Protein-Coupled Receptors
Adenosine
Real-Time Polymerase Chain Reaction
Membrane Proteins
Oxygen
Kidney

Keywords

  • Adenosine receptors
  • Neovascularization
  • Retinopathy
  • Ribozymes

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Afzal, A., Shaw, L. C., Caballero, S., Spoerri, P. E., Lewin, A. S., Zeng, D., ... Grant, M. B. (2003). Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA. Circulation Research, 93(6), 500-506. https://doi.org/10.1161/01.RES.0000091260.78959.BC

Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA. / Afzal, A.; Shaw, L. C.; Caballero, S.; Spoerri, P. E.; Lewin, A. S.; Zeng, D.; Belardinelli, L.; Grant, M. B.

In: Circulation Research, Vol. 93, No. 6, 19.09.2003, p. 500-506.

Research output: Contribution to journalArticle

Afzal, A, Shaw, LC, Caballero, S, Spoerri, PE, Lewin, AS, Zeng, D, Belardinelli, L & Grant, MB 2003, 'Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA', Circulation Research, vol. 93, no. 6, pp. 500-506. https://doi.org/10.1161/01.RES.0000091260.78959.BC
Afzal, A. ; Shaw, L. C. ; Caballero, S. ; Spoerri, P. E. ; Lewin, A. S. ; Zeng, D. ; Belardinelli, L. ; Grant, M. B. / Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA. In: Circulation Research. 2003 ; Vol. 93, No. 6. pp. 500-506.
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