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(Circulation Research. 2004;95:814.)
© 2004 American Heart Association, Inc.

Integrative Physiology

Targeted Disruption of cd73/Ecto-5'-Nucleotidase Alters Thromboregulation and Augments Vascular Inflammatory Response

Patrycja Koszalka, Burcin Özüyaman, Yuqing Huo, Alma Zernecke, Ulrich Flögel, Norbert Braun, Anja Buchheiser, Ulrich K.M. Decking, Michael L. Smith, Jean Sévigny, Adrian Gear, Artur-Aron Weber, Andrei Molojavyi, Zhaoping Ding, Christian Weber, Klaus Ley, Herbert Zimmermann, Axel Gödecke, Jürgen Schrader

From the Department of Cardiovascular Physiology (P.K., B.Ö., U.F., A.B., U.K.M.D., A.M., Z.D., A. Gödecke, J. Schrader), Heinrich-Heine-University, Düsseldorf, Germany; Biozentrum der J.W. Goethe-Universität, AK Neurochemie, Zoologisches Institut (N.B., H.Z.), Frankfurt am Main, Germany; Departments of Molecular Cardiovascular Research and Cardiology (A.Z., C.W.), University of Technology, Aachen, Germany; Cardiovascular Research Center and Department of Biomedical Engineering (Y.H., M.L.S., K.L.), Charlottesville, Va; Department of Biochemistry and Molecular Genetics (A. Gear), University of Virginia Health Sciences Center, Charlottesville, Va; Department of Pharmacology (A.A.W.), Heinrich-Heine-University, Düseldorf, Germany; and Centre de Recherche en Rhumatologie et Immunologie (J. Sévigny), Sainte-Foy (Québec), Canada. The present address for P.K. is the Department of Cell Biology, Intercollegiate Faculty of Biotechnology, Medical University of Gdask, Poland.

Correspondence to Juergen Schrader, MD, Heinrich-Heine-University Duesseldorf, Department of Cardiovascular Physiology, Moorenstrasse 5, 40225 Duesseldorf, Germany. E-mail schrader{at}uni-duesseldorf.de

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5'-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (–14%; P<0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl₃) was significantly reduced by 20% in cd73^–/– mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73^–/– mice (–35%). In vivo platelet cAMP levels were 0.96±0.46 in WT versus 0.68±0.27 pmol/10⁶ cells in cd73^–/– mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 µmol/L) was undistinguishable between the two strains. In the cremaster model of ischemia–reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73^–/– compared with WT littermates (WT 98% versus cd73^–/– 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo–perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73^–/– mice (P<0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.

Key Words: transgenic mice • adenosine • ecto-5'-nucleotidase • vascular inflammation • thrombosis

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