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-Although CD73/ecto-5Ј-nucleotidase has been implicated in maintaining vasoprotection, its role in regulating endothelial adhesion molecule or inflammatory monocyte recruitment (eg, in the context of vascular injury) remains to be defined. Methods and Results-Compared with wild-type mice, CD73-deficient (CD73 Ϫ/Ϫ ) mice exhibit increased luminal staining and protein and transcript expression for vascular cell adhesion molecule (VCAM)-1 in carotid arteries. In vitro, aortic endothelial cells (ECs) from CD73 Ϫ/Ϫ mice display an upregulation of mRNA and protein expression of VCAM-1, associated with increased nuclear factor (NF)-B activity, as determined by chromatin cross-linking and immunoprecipitation or quantitative p65 binding assays. CD73Ϫ/Ϫ ECs and carotid arteries perfused ex vivo supported increased monocyte arrest under flow conditions, which was mediated by ␣ 4␤1 integrin. After wire injury of carotid arteries, CD73 expression and activity were upregulated in wild-type mice, whereas neointimal plaque formation and macrophage content were increased in CD73 Ϫ/Ϫ mice versus wild-type mice, concomitant with elevated NF-B activation, luminal VCAM-1 expression, and soluble VCAM-1 concentrations. In contrast, reconstitution of wild-type mice with CD73

To investigate the role of nitric oxide in controlling endothelial progenitor (EPC) and hematopoietic stem cell (HSC) mobilization, wild-type mice, L-NAME treated WT and eNOS-/- mice received either PBS or G-CSF for 5 days. Under unstimulated conditions bone marrow of either L-NAME treated WT and eNOS-/- mice, representing acute and chronic NO-deficiency, showed higher CD34(+)Flk-I+ EPC numbers compared to their WT littermates. Furthermore, CD34(+)Flk-I+ progenitors under NO-deficient conditions showed a higher cell turn over since the proliferation and apoptosis activity under in vivo as well as in vitro conditions were enhanced. In line with this finding bone marrow derived EPC differentiation towards endothelial cells was modulated in an NO-dependent manner. Administration of G-CSF resulted in an increase of EPC within the bone marrow of WT animals with a consecutive release of these cells into the peripheral circulation. Under NO-deficient conditions G-CSF failed to increase EPC numbers. In contrast, the HSC population c-kit(+)Lin- was not influenced by nitric oxide. Thus, NO differentially supports the mobilization of the endothelial committed progenitor subpopulation in bone marrow but does not have an effect on HSC in vivo.

No abstract

Endothelial NO synthase (eNOS) expressed in the vascular endothelium or formed within platelets was postulated to inhibit platelet activation and aggregation. We have assessed the role of eNOS in platelet aggregation in vitro and in vivo by comparison of WT and eNOS-/- mice. Aggregometer studies revealed that collagen over a concentration range of 0.36-10 microg aggregated WT and eNOS-/- platelets to the same extent (10 microg: WT 86.7+/-4.7%, eNOS-/- 91+/-12%, n=6). Collagen treatment did not result in a significant increase in cGMP formation and VASP phosphorylation. Thrombin-induced P-selectin surface expression was unchanged in eNOS-/-platelets. In line with these findings no eNOS protein was detectable within the platelets of WT mice. In vivo, bleeding time after tail tip resection tended to be shorter in eNOS/- mice (WT: 116+/-35 s; eNOS-/- 109+/-37 s, n.s). Similarly, time to occlusion of the A.carotis after focal induction of thrombosis was 501+/-76 s (WT) and 457+/-95 s (eNOS-/-) (n.s.). These data demonstrate that eNOS-deficiency minimally affects platelet aggregation and is not associated with accelerated arterial thrombosis in vivo. Thus, in the mouse endothelial NO synthase does not play a major role in the autocrine modulation of platelet function and in thrombosis of conduit vessels in vivo.