Published online before print September 9, 2004, doi: 10.1161/01.RES.0000144796.82787.6f
© 2004 American Heart Association, Inc.
Integrative Physiology |
Targeted Disruption of cd73/Ecto-5'-Nucleotidase Alters Thromboregulation and Augments Vascular Inflammatory Response
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 Gda
sk, 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
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Abstract |
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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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Introduction |
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CD73/ecto-5'-nucleotidase, a 70-kDa glycosylphosphatidylinositol (GPI)-anchored cell surface molecule, is expressed on the vascular endothelium and catalyzes the extracellular conversion of 5'-AMP to adenosine.1,2 CD73 is the final step of the extracellular nucleotide breakdown cascade that also involves membrane-associated CD39/ATP-diphosphohydrolase.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: A1, A2A, A2B, and A3, each of which operates via different intracellular signaling mechanisms and exhibits distinct patterns of tissue distribution.5 In human neutrophils, adenosine A1 and A2 receptor occupancy mediate opposing roles for adenosine in inflammation: A1 activation is proinflammatory, whereas the A2 receptor plays an anti-inflammatory role.6 A2 receptor activation inhibits the neutrophil oxidative burst, whereas the A3 receptor inhibits neutrophil degranulation7 and may play an important role in inflammation by inhibiting eosinophil migration.8 Recently, deletion of the A2A receptor in transgenic mice revealed that this receptor is critical for the limitation and termination of tissue-specific and systemic inflammatory responses.9 Similar to neutrophils, platelet A2A receptors are well known to inhibit ADP-induced aggregation5,10 via a cAMP-mediated process; however, whether and under which conditions this is functionally relevant in vivo has not been explored.
The quantitatively most important source of adenosine in most organs under well-oxygenated conditions is 5'-AMP hydrolyzed by cytosolic 5'-nucleotidase N-I.11,12 It was estimated that the coronary endothelium only contributes 
5% to total cardiac adenosine production11 so that >90% of the overall production occurs intracellularly. Thus, it is unclear whether the in vivo production of adenosine by extracellular adenine nucleotide breakdown involving CD73 in a final step is quantitatively sufficient in vivo to mediate the antiaggregatory and anti-inflammatory actions demonstrated previously in pharmacological experiments.
Theoretically, changes in the level of extracellular adenosine caused by changes in substrate supply to CD73 may represent an essential mechanism for thromboregulation and inflammation in the vasculature. However, there are other potentially redundant properties of the vascular endothelium that influence the activity of platelets and neutrophils13 such as NO and prostaglandins, which may compete with CD73-derived adenosine. Therefore, to verify the importance of CD73 in vascular control, we generated mice deficient in CD73 and characterized their hemodynamic, thromboregulatory and inflammatory phenotype.
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Materials and Methods |
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Generation of Mutant Mice
A mouse 129Sv genomic library in a vector Lambda DASH II was constructed in our laboratory and screened using as a probe 1.8 kb EcoRI fragment of rat cd73 gene spanning exon 2.14 A detailed description of the construction of targeting vector and generation of mutant mice is available in the online data supplement at http://circres.ahajournals.org.
Mice
Mice were bred at the Tierversuchsanlage of the Heinrich-Heine-Universität in Düsseldorf, Germany. Animal experiments were performed in accordance with the national guidelines on animal care. Mice 2 to 3 months of age were used for this study. Animals received care in accordance with the European Convention on Animal Care, and the protocol was approved by the Bezirksregierung Düsseldorf.
RT-PCR Analysis of Mutant Mice
Total RNA was isolated from mouse tissues of the various genotypes using RNeasy Mini Kits (Qiagen). Changes in cd73 cDNA were identified by PCR using primer 5 (5'-CCATCTGGTTCACCGTTTAC-3'), designed from the sequence of exon 1, and primer 6 (5'-GGGCGATGATCTTATTCACAT-3'), from the sequence of exon 3 of the cd73 gene.
Preparation of Antibodies and Western Blot Analysis
Anti-rat liver ecto-5'-nucleotidase antibody was raised in rabbits by direct injection of the encoding cDNA ligated into the plasmid pcDNA3.14 Western blot analysis was performed under reducing conditions using the enhanced chemiluminescence system (Amersham Biosciences).
Biochemical Analysis of Ecto-5'-Nucleotidase Activity
Ecto-5'-nucleotidase was extracted from hearts and livers of knockout and wild-type (WT) mice and purified using concanavalin A(ConA)-Sepharose (Sigma) as described previously.15 Activity of 5'-nucleotidase was determined by analysis of phosphate with 500 µmol/L AMP (Roche Applied Science) as a substrate (pH 7.4).16 To block alkaline phosphatase (AP) activity, 5 mmol/L levamisole (Sigma) was added.
Enzyme Histochemistry
For localization of ecto-5'-nucleotidase activity, frozen sections and a lead phosphate method (pH 7.4) were applied as described previously.14 A detailed description is provided in the online data supplement.
Langendorff Heart
Preparation of murine hearts and retrograde perfusion at constant pressure of 100 mm Hg were performed as described.17 For quantification of AMP hydrolysis in vivo, 50 µmol/L 1,N6-ethenoadenosine 5'-monophosphate (e-AMP) was infused, and the coronary venous effluent was analyzed on the basis of a protocol published previously.18
Cardiac Function In Vivo
Hemodynamic parameters were analyzed as described previously.17 In brief, mice were anesthetized with urethane, kept at 37°C, and after preparation of the left common carotid artery, a Millar 1.4 French pressure-volume catheter was advanced into the left ventricle.
Tail Bleeding and Time to Occlusion
Tail bleeding times were measured in anesthetized mice (urethane 1.5 g/kg IP) after resection of 0.5 cm of the tail tip. Tails were submerged in 37°C warm saline, and the time to cessation of blood flow was recorded. In a separate set of experiments, the carotid artery model for arterial thrombus formation was used.19 A vascular lesion was induced by covering the carotid artery with a 7.5% solution of FeCl3 and the cessation of blood flow was measured by an ultrasound flow probe (Transsonics).
Platelet cAMP/cGMP
Fresh citrated blood was obtained by puncturing the inferior vena cava. Blood was immediately resuspended with the unspecific phosphodiesterase inhibitor isopbutylmethylxanthine (IBMX; 0.5 mmol/L). Platelet-rich plasma (300 µL) was deproteinized by addition of 60 µL 25% HClO4 and neutralized with 75 µL 2 mol/L K2CO3. cAMP and cGMP were determined by radioimmunoassay.
Intravital Microscopy
Mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg; Ketalar, Parke-Davis), xylazine (0.05 mg/kg), and atropine (0.1 mg/kg; Elkins-Sinn). Animals were kept at 37°C throughout the experiment with a heating pad. The cremaster muscle was externalized over a Plexiglas observation platform and pinned in place as described previously.20
Ex Vivo Perfusion of Murine Carotid Arteries
Carotid arteries from cd73–/– mice and WT mice were isolated for ex vivo perfusion as described.21,22 As an established model, monocytic Mono Mac 6 cells (106/mL) labeled with calcein-acetoxymethyl (Molecular Probes) were perfused at 7 µL per minute, and adhesive interactions with the vessel wall (arrest, rolling) were recorded using stroboscopic epifluorescence illumination (Drelloscop 250; Drello). Firmly adherent monocytes were counted after 10 minutes of perfusion.
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Results |
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The cd73 gene of the mouse was disrupted by homologous recombination and activation of the Cre-loxP system (Figure 1). WT, heterozygous, and homozygous offspring from interbreeding heterozygote mice followed approximately Mendelian frequencies of 0.22: 0.54: 0.23 (n=175). There were no abnormalities in development and reproductive capacity in cd73–/– mice.
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Southern blot analysis of the genomic structures of mice of all three cd73 genotypes of the F2 offspring revealed that the cd73 locus was properly targeted (Figure 1). RT-PCR analysis of the CD73 transcripts showed that deletion of exon 2 resulted in a shortened mRNA expression at similar levels as the WT transcript (Figure 2a). Because of a frame shift at the new exon 1 to 3 junction, translation terminates after the first exon, which leads to the loss of most the amino acid sequence, including the active site of the protein and its C-terminal domain needed for GPI–anchor linkage to the membrane.
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Consistently, Western blot analysis demonstrated the absence of protein within ConA-purified protein fractions derived from membrane fractions of heart and liver tissue (Figure 2b). The biochemical activity of AMP hydrolysis as a consequence of gene deletion was tested in the ConA-purified protein fractions and showed a >50-fold decrease in the liver and 10-fold decrease in the heart of cd73–/– mice compared with their WT littermates (Figure 2c). The small residual activity within the heart of cd73–/– mice was further reduced to background activity in the presence of levamisole, an inhibitor of AP23 (Figure 2c).
Activity of CD73 at the cellular level was identified using histochemical methods. In WT animals, a strong reaction product for AMP hydrolysis was identified in the endothelium of the carotid (Figure 3a, left) and coronary artery (Figure 3a, right). In cd73–/– mice, the enzyme reaction was completely abolished. The same result was obtained for the aorta and the femoral artery and vein (data not shown). As has been described earlier,24 CD73 was only partially associated with the microcirculatory bed of the myocardium. In WT mice, strong AMP hydrolysis was associated with myocardial arterioles and capillaries (Figure 3b, top left). In cd73–/– mice, AMP hydrolysis was reduced but still associated with microvasculature (Figure 3b, top right), which is attributable to the presence of the nonspecific form of AP. Accordingly, in the presence of levamisole, enzyme histochemical staining was reduced but not eliminated in WT mice (Figure 3b, bottom left). In cd73–/– mice, levamisole abolished microvascular AMPase activity (Figure 3b, bottom right). In addition, myocardial background staining was eliminated in cd73–/– mice in the presence of levamisole.
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Intensified staining using the azo dye method and alkaline pH (8.7) revealed that only part of the myocardial capillaries expresses AP. This is clearly demonstrated using double labeling for the endothelial marker IB4 (Figure 3c, left), which often reveals discontinuation of AP activity on the identical microvessel. In addition, double histochemical staining for CD73, pH 7.4, and AP (pH 8.7) in WT animals revealed a separate expression of the two enzymes in the microvasculature of the heart muscle (Figure 3c, right). In few cases, a transition of CD73 to AP-positive microvessels could be observed (data not shown).
In vivo analysis of contractile and hemodynamic parameters in mice using a conductance catheter that was advanced through the carotid artery to the heart revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between WT and cd73–/– mice (Table). Also, animal weight and heart-to-body weight index showed no differences (data not shown). However, we found basal coronary flow in the isolated perfused heart to be significantly lower (–14%) in the mutant compared with the WT (Table).
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The reactive hyperemic flow response after 30 seconds of coronary flow occlusion was not different between WT and cd73–/– mice (data not shown), indicating a predominant role of factors other than extracellularly formed adenosine. Further functional analysis of the isolated perfused heart revealed that the dose-response curve for adenosine-applied intracoronary (1 to 200 µmol/L) revealed no differences between WT and cd73–/– mice (data not shown), suggesting unaltered A2A receptor density/coupling. However, the dose-response curve for AMP was shifted to the right by about one order of magnitude, demonstrating that the dilatory effect of AMP requires hydrolysis to vasoactive adenosine (Figure 4). This result could be confirmed when WT hearts were perfused with 50 µmol/L 
,ß-methylene ADP, an inhibitor of CD73, leading to a decreased AMP response. Consistent with this finding, perfusion of hearts with fluorescent etheno-AMP resulted in hydrolysis of 24% of this precursor to etheno-adenosine in the WT during single passage through the heart, whereas the respective value in cd73–/– mice was only 9% (Figure 4). The residual AMPase activity in cd73–/– mice is most likely attributable to AP.
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Peripheral blood platelet counts in cd73–/– mice (449±95x103/µL) were similar to those in WT controls (413±47x103/µL). Further evaluation of other relevant hematological parameters revealed no abnormalities in mutant mice.
To evaluate the role of CD73 in platelet thrombosis formation in vivo, carotid arteries of WT and cd73-deficient mice were subjected to free radical injury using FeCl3. In this model, time to vessel occlusion as measured with a sensitive flow probe was significantly shorter (–20%) in cd73–/– mice (Figure 5a). Bleeding time after tail tip resection was reduced by 35% in cd73–/– mice but did not reach the level of significance because of the large SE associated with this method (Figure 5b). Platelet cAMP but not cGMP was significantly lower in cd73–/– mice (Figure 5c), suggesting that under in vivo conditions, the concentration of plasma adenosine was reduced in cd73–/– mice. We also were unable to detect CD73 on platelets by flow cytometry using mutant platelets as negative controls. Under in vitro conditions, platelet aggregation in response to ADP (dose range 0.05 to 10 µmol/L) was undistinguishable between platelets isolated from cd73–/– mice and WT littermates (data not shown). This is not surprising given the turnover of cellular cAMP and the short half life of plasma adenosine.25
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To determine the role of endogenous adenosine produced through the CD73 pathway in the vascular inflammatory response, mouse cremaster muscles were subjected to ischemia–reperfusion injury as described.26 As shown in Figure 6, ischemia (30 minutes) followed by reperfusion significantly increased the number of adherent leukocytes in cd73–/– mice. In WT littermates, ischemia–reperfusion increased the number of adherent leukocytes by 98%, whereas in cd73 mutants, the respective increase was 245% (P<0.005). Even under basal unstressed conditions, the number of adherent leukocytes tended to be higher in cd73–/– mice compared with that in WT but did not reach the level of significance. Maximal increase in adherent leukocytes occurred between 10 and 15 minutes after the onset of reperfusion. There was no change of blood flow in microvessels of cd73–/– versus control mice. Average centerline velocity was 2.1±0.8 mm per second in cd73+/+ mice and 1.9±0.9 mm per second in cd73–/– mutants (mean±SD).
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Because a strong activity of CD73 was found in carotid and coronary arteries, the ex vivo perfusion of carotid arteries was performed to further elaborate on a constitutive role of CD73 in anti-inflammatory vasoprotection. Whereas the background adhesion of monocytes perfused in carotid arteries of WT mice was negligible, perfusion of monocytes in carotid arteries of cd73–/– mice resulted in a remarkable (> 6-fold) and significant (P<0.05) increase in accumulation after 10 minutes of perfusion (Figure 7). In contrast to monocyte accumulation on early atherosclerotic endothelium in carotid arteries of apoE–/– mice, where arrest occurs preferentially at the lesion-prone predeliction site of the bifurcation, enhanced arrest in cd73–/– was also seen in other segments of the perfused artery.21 Rolling interactions of monocytes analyzed over 10 minutes were not significantly altered in cd73–/– arteries (data not shown).
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Discussion |
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In the present study, we obtained convincing evidence for successful gene targeting and generation of cd73-deficient mice by Southern blot analysis (Figure 1b), RT-PCR (Figure 2a), and Western blot analysis (Figure 2b) of selected tissues known to exhibit RNA transcripts and ecto-5'-nucleotidase activity (Figure 2c). We deleted exon 2 encoding the "Phosphoesterase Signature Motive," a sequence pattern involved in phosphoester bond cleavage conserved in a wide variety of phosphoesterases.27 Use of the Cre-loxP system involving 3 loxP sites (Figure 1) enabled us to generate, in one experiment, animals containing constitutive and conditional knockout of the cd73 gene, the latter being important in future studies on tissue-specific cd73 deletion.28 Our approach also enabled the effective removal of a selectable marker cassette, thereby eliminating the possibility of influencing the expression of the neighboring genes, which can lead to unexpected phenotypes in "knockout" mice.29
AMP-hydrolyzing activity of cell membranes comprises CD73 and AP activity, the former being a high-affinity enzyme and the latter being of low affinity.30 Thus, ecto-5'-nucleotidase is expected to be more efficient than AP at producing adenosine at physiological AMP and pH. CD73 is a prominent enzyme in the heart and liver under Vmax conditions (Figure 2c), whereas in the isolated perfused heart, AMP hydrolyzing activity attributable to CD73 amounts to 60% of total hydrolytic activity during single passage through the coronary vasculature (Figure 4b). Histochemical data revealed CD73 to be the predominant AMPase associated with the vascular endothelium of large conduit vessels such as the aorta, carotid, and coronary artery with no measurable AP. However, at the coronary microcirculation, there is substantial local heterogeneity (Figure 3c).
Adenosine is a primordial signaling molecule that has evolved to modulate a multitude of physiological responses in all mammalian tissues.5 Pharmacological activation of A2A receptors through Gs activation causes vasodilatation of coronary arteries and produces a constellation of responses that can be classified as anti-inflammatory.31 The present study is the first to demonstrate that adenosine generated by the extracellular hydrolysis of adenine nucleotides by action of CD73 is an important endogenous pathway to modulate the inflammatory vascular response. After a brief period of ischemia–reperfusion, we observed significantly increased leukocyte adherence to the vascular endothelium only in the cd73 mutant (Figure 6). Thus, under physiological conditions with unperturbed extracellular hydrolysis of AMP, adenosine generated by this pathway appears to be quantitatively sufficient to keep circulating leukocytes in a nonactivated state.
Modulation of leukocyte adhesion by CD73 generating adenosine under physiological conditions was corroborated by findings that monocyte adhesion was constitutively increased in carotid arteries of cd73–/– mice, whereas arteries of WT mice displaying strong CD73 activity supported only little arrest. The absence of paracrine effects by adenosine on both endothelial cells and monocytes may thus account for the mechanism of enhanced monocyte accumulation in cd73–/– mice. The adhesion of monocytes on endothelium in carotid arteries of apoE–/– mice has been shown to depend on interactions of 4ß1 integrin (very-late antigen-4 [VLA-4]) with vascular cell adhesion molecule (VCAM)-1 and is triggered by the murine interleukin-8 (IL-8) ortholog keratinocyte-derived chemokine.21,22 Conversely, adenosine has been shown to dose-dependently reduce the expression of VCAM-1 and the arrest chemokine IL-8 by activated endothelial cells,32 providing one possible explanation for a suppressive interference with this crucial axis by endogenous adenosine. Furthermore, neutrophil–endothelial cell adhesion can be attenuated by elevating intracellular cAMP. This effect can be attributed to inhibition of Mß2 integrin upregulation by formyl-methionyl-leucyl-phenylalanine. Prevention by adenosine deaminase implicates endogenous adenosine as a comodulator of inhibition.33 In neutrophils, adenosine binding to A2A receptors has been shown to counteract the stimulation of VLA-4 expression and binding to VCAM-1 by chemoattractants via a cAMP-mediated pathway.34 This may not only explain the increase in leukocyte attachment observed after ischemia—reperfusion; because VLA-4 is the primary adhesive receptor in monocytes, it is conceivable that chemokine-triggered VLA-4 adhesiveness mediating monocyte arrest in arteries may also be affected by endothelial-derived adenosine. The generation of adenosine by CD73 may thus provide a general vasoprotection against leukocyte adhesion.
Blood platelets maintain vascular integrity and promote primary and secondary hemostasis after interruption of vessel continuity. They undergo profound shape changes in the presence of ADP mediated by cell surface P2Y receptors. The proaggregatory action of ADP is sensitively inhibited by adenosine and mediated by antiaggregatory A2A receptors.5 It has been postulated for quite some time that adenosine formed at the endothelial platelet interface may limit thrombus formation.35,36 Our study provides experimental evidence that this hypothesis is valid. We found that the time to carotid occlusion after FeCl3 application was reduced by 20% in cd73 mutants (Figure 5). However, we found no differences in the aggregatory response of platelets to ADP in vitro, which is consistent with no measurable CD73 activity on mouse platelets and the extremely short plasma half life of adenosine.25 The decreased platelet cAMP combined with unchanged cGMP in cd73 mutants is most likely a consequence of a lower in vivo adenosine level in the immediate milieu of the vascular endothelium.
For the extracellular cascade from ATP to adenosine to be functionally relevant during thromboregulation and inflammation, an intact endothelium capable of releasing adenine nucleotides on external stimuli is required. Cellular ATP release may involve transport through ATP-binding cassette proteins, vesicular release,37 and connexin hemichannels in endothelial cells.38 Ischemia–reperfusion but not hypoxia was shown by us to release ATP from the coronary endothelium,39 and more recently, an increased flow was reported to release ATP from endothelial cells in pulmonary circulation.40 Thus, there appears to be a basal flux of purines through this extracellular nucleotide cascade that can be further augmented by ATP/ADP release from activated platelets and neutrophils under pathophysiological conditions.
It is generally assumed that during hypoxia, the intracellular formation from parenchymal cells dominates and may be responsible for the phenomenon of metabolic coronary vasodilation. Here we show that in the mouse, adenosine formed by the extracellular nucleotide cascade is involved in setting basal coronary vascular tone postulated previously by pharmacological evidence to be adenosine independent.41 Thus, CD73-derived adenosine, most likely attributable to short diffusion distances, can directly influence vascular smooth muscle tone. How this finding translates to the blood perfused heart in vivo is difficult to ascertain at present. We found blood pressure and cardiac output to be normal in cd73 mutants, suggesting that in vivo at the given high oxygen extraction rate of the heart, the loss of CD73-derived adenosine may have been compensated by other factors.
Whereas ATP acts on extracellular purinergic P2 receptors, its degradation to adenosine activates P1 receptors. Interruption of the cascade from ATP to adenosine thus converts a P1 to a P2 environment at the cell surface irrespective of the site of blockade. The recently published disruption of the cd39/ATP diphosphohydrolase3 and the cd73 mutant reported here interrupt the extracellular nucleotide cascade, yet at different stages. However, the formation of adenosine should be equally reduced in both mutants, and given our present findings, changes in adenosine are likely to have importantly contributed to the reported phenotype in the former model. It also should be noted that lack of elimination of nucleotides in the cd73 mutant may have altered local levels of upstream ADP and ATP, and thereby could have contributed to inflammation.
In summary, the present study provides the first evidence that the terminal step of the extracellular adenine nucleotide cascade on endothelial cells, which converts AMP to adenosine, modulates coronary vascular tone and inhibits platelet function and leukocyte adhesion. This implicates the CD73-mediated pathway as a key innate mechanism to attenuate tissue inflammation.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft (SFB612 and We 1913/5-1). B.Ö. is a recipient of a grant by the German Cardiac Society. We wish to thank Barbara Patzer, Susanne Küsters, and Andrea Quadflieg for excellent technical assistance and animal care. This work is dedicated to Prof Eckehart Gerlach, a pioneer of adenosine research.
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Footnotes |
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Original received June 25, 2004; revision received August 12, 2004; accepted September 1, 2004.
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References |
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