Abstract-To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5Ј-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 (Ϫ14%; PϽ0.05) 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 (FeCl 3 ) was significantly reduced by 20% in cd73 Ϫ/Ϫ mice (PϽ0.05). Bleeding time after tail tip resection tended to be shorter in cd73mice (Ϫ35%). In vivo platelet cAMP levels were 0.96Ϯ0.46 in WT versus 0.68Ϯ0.27 pmol/10 6 cells in cd73 Ϫ/Ϫ mice (PϽ0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 mol/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 Ϫ/Ϫ compared with WT littermates (WT 98% versus cd73 Ϫ/Ϫ 245%; PϽ0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73 Ϫ/Ϫ mice (PϽ0.05). 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 Ⅲ adenosine Ⅲ ecto-5Ј-nucleotidase Ⅲ vascular inflammation Ⅲ thrombosis C D73/ecto-5Ј-nucleotidase, a 70-kDa glycosylphosphatidylinositol (GPI)-anchored cell surface molecule, is expressed on the vascular endothelium and catalyzes the extracellular conversion of 5Ј-AMP to adenosine. 1,2 CD73 is the final step of the extracellular nucleotide breakdown cascade that also involves membrane-associated CD39/ATPdiphosphohydrolase. 3 The product of CD73 is adenosine, a purine nucleoside that has been implicated in many physiological and pathophysiological events. 4 There are four known G-coupled adenosine receptors: A 1 , A 2A , A 2B , and A 3 , each of which operates via different intracellular signaling mechanisms and exhibits distinct patterns of tissue distribution. 5 In human neutrophils, adenosine A 1 and A 2 receptor occupancy mediate opposing roles for adenosine in inflammation: A 1 activation is proinflammatory, whereas the A 2 receptor plays an anti-inflammatory role. 6 A 2 receptor activation inhibits the neutrophil oxidative burst, whereas the A 3 receptor inhibits neutrophil degranulation 7 and may play an important role in inflammation by inhibiting eosinophil migration. 8 Recently, deletion of the A 2A receptor in transgenic mice revealed that this receptor is critical for the limitation a...
Background-Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. Methods and Results-Four-week-old apolipoprotein E-deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. Conclusions-The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis. (Circulation. 2010;122:2313-2322.)Key Words: atherosclerosis Ⅲ inflammation Ⅲ glycocalyx Ⅲ hyaluronan H yaluronan is a ubiquitous constituent of the extracellular matrix. The synthesis is mediated through 3 hyaluronan synthase (HAS) isoforms (HAS1, HAS2, and HAS3) that assemble UDP-glucuronic acid and UDP-N-glucosamine at the plasma membrane, forming a high-molecular-weight glycosaminoglycan chain. 1 Hyaluronan is unbranched and is not further modified, in contrast to sulfated glycosaminoglycans such as heparan sulfate. 2 Extensive research on the hyaluronan system in tumor biology, 2 reproductive biology, 3 lung injury, 4 and cardiovascular pathology 5,6 has contributed to an understanding of the physiological and pathophysiological role of hyaluronan in vitro and in vivo. Clinical Perspective on p 2322In the healthy arterial vessel wall, hyaluronan is positioned at 2 strategic positions: the endothelial glycocalyx and the adventitia. However, during atherosclerosis, hyaluronan is produced by activated vascular smooth muscle cells (VSMC) in the neointima. Extensive evidence from studies on atherosclerosis and restenosis shows that hyaluronan promotes VSMC proliferation and migration and that hyaluronan accu...
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