CD73 Promotes Age-Dependent Accretion of Atherosclerosis.

OBJECTIVE: CD73 is an ectonucleotidase which catalyzes the conversion of AMP (adenosine monophosphate) to adenosine. Adenosine has been shown to be anti-inflammatory and vasorelaxant. The impact of ectonucleotidases on age-dependent atherosclerosis remains unclear. Our aim was to investigate the role of CD73 in age-dependent accumulation of atherosclerosis. Approach and results: Mice doubly deficient in CD73 and ApoE (apolipoprotein E; (cd73-/-/apoE-/-) were generated, and the extent of aortic atherosclerotic plaque was compared with apoE-/- controls at 12, 20, 32, and 52 weeks. By 12 weeks of age, cd73-/-/apoE-/- mice exhibited a significant increase in plaque (1.4±0.5% of the total vessel surface versus 0.4±0.1% in apoE-/- controls, P<0.005). By 20 weeks of age, this difference disappeared (2.9±0.4% versus 3.3±0.7%). A significant reversal in phenotype emerged at 32 weeks (9.8±1.2% versus 18.3±1.4%; P<0.0001) and persisted at the 52 week timepoint (22.4±2.1% versus 37.0±2.1%; P<0.0001). The inflammatory response to aging was found to be comparable between cd73-/-/apoE-/- mice and apoE-/- controls. A reduction in lipolysis in CD73 competent mice was observed, even with similar plasma lipid levels (cd73-/-/apoE-/- versus apoE-/- at 12 weeks [16.2±0.7 versus 9.5±1.4 nmol glycerol/well], 32 weeks [24.1±1.5 versus 7.4±0.4 nmol/well], and 52 weeks [13.8±0.62 versus 12.7±2.0 nmol/well], P<0.001). CONCLUSIONS: At early time points, CD73 exerts a subtle antiatherosclerotic influence, but with age, the pattern reverses, and the presence of CD73 promoted suppression of lipid catabolism.

Tissue-resident ecto-5' nucleotidase (CD73) regulates leukocyte trafficking in the ischemic brain.

Ectoenzymes expressed on the surface of vascular cells and leukocytes modulate the ambient nucleotide milieu. CD73 is an ecto-5' nucleotidase that catalyzes the terminal phosphohydrolysis of AMP and resides in the brain on glial cells, cells of the choroid plexus, and leukocytes. Though CD73 tightens epithelial barriers, its role in the ischemic brain remains undefined. When subjected to photothrombotic arterial occlusion, CD73(-/-) mice exhibited significantly larger (49%) cerebral infarct volumes than wild-type mice, with concordant increases in local accumulation of leukocyte subsets (neutrophils, T lymphocytes, macrophages, and microglia). CD73(-/-) mice were rescued from ischemic neurologic injury by soluble 5'-nucleotidase. In situ, CD73(-/-) macrophages upregulated expression of costimulatory molecules far more than wild-type macrophages, with a sharp increase of the CD80/CD86 ratio. To define the CD73-bearing cells responsible for ischemic cerebroprotection, mice were subjected to irradiative myeloablation, marrow reconstitution, and then stroke following engraftment. Chimeric mice lacking CD73 in tissue had larger cerebral infarct volumes and more tissue leukosequestration than did mice lacking CD73 on circulating cells. These data show a cardinal role for CD73 in suppressing ischemic tissue leukosequestration. This underscores a critical role for CD73 as a modulator of brain inflammation and immune function.

Ecto-5'-nucleotidase (CD73) attenuates allograft airway rejection through adenosine 2A receptor stimulation.

There are multiple drivers of leukocyte recruitment in lung allografts that contribute to lymphocytic bronchitis (LB) and bronchiolitis obliterans (BO). The innate mechanisms driving (or inhibiting) leukocyte trafficking to allografts remain incompletely understood. This study tested the hypothesis that CD73 (ecto-5'nucleotidase), an enzyme that catalyzes the conversion of AMP to adenosine, is a critical negative regulator of LB and BO. Implantation of tracheal allografts from wild type (WT) mice into CD73(-/-) recipients revealed a striking increase in airway luminal obliteration at 7 d (62 +/- 4% and 47 +/- 5% for CD73(-/-) and WT allograft recipients, respectively; p = 0.046). There was also a concordant increase in CD3(+) lymphocytic infiltration (523 +/- 41 cells and 313 +/- 43 cells for CD73(-/-) and WT allograft recipients, respectively; p = 0.013). Because real-time PCR revealed a 43-fold upregulation of mRNA for the adenosine A2A receptor (A2AR) in WT allografts compared with WT isografts (p = 0.032), additional experiments were performed to determine whether the protective effect of CD73 was due to generation of adenosine and its stimulation of the A2AR. Treatment of WT recipients with an A2AR agonist significantly reduced CD3(+) lymphocyte infiltration and airway luminal obliteration; similar treatment of CD73(-/-) recipients rescued them from LB and airway obliteration. These data implicate CD73 acting through adenosine generation and its stimulation of the A2AR as a critical negative modulator of lymphocyte recruitment into airway allografts. The CD73/adenosine axis might be a new therapeutic target to prevent BO.

Ecto-5' nucleotidase (CD73)-mediated adenosine generation and signaling in murine cardiac allograft vasculopathy.

Ecto-5'-nucleotidase (CD73) catalyzes the terminal phosphohydrolysis of 5'-adenosine monophosphate and is widely expressed on endothelial cells where it regulates barrier function. Because it is also expressed on lymphocytes, we hypothesized that it modulates vascular immune regulation under homeostatic conditions and dysregulation under stress conditions such as cardiac allotransplantation. In a heterotopic cardiac allotransplantation model, CD73 deficiency in either donors or recipients resulted in decreased graft survival and the development of cardiac allograft vasculopathy, suggesting a contribution of CD73 on both graft-resident and circulating cells in vasculopathy pathogenesis. Vascular perturbations incited by lack of CD73 included loss of graft barrier function and diminished graft expression of the A(2B) adenosine receptor (A(2B)AR), with a concordant exacerbation of the acute inflammatory and immune responses. The importance of CD73 in modulating endothelial-lymphocyte interaction was further demonstrated in allomismatched in vitro coculture experiments. Either genetic deletion or pharmacological blockade of CD73 increased transendothelial lymphocyte migration and inflammatory responses, suggesting that CD73 plays a critical role to suppress transendothelial leukocyte trafficking through its enzymatic activity. In addition, antagonism of A(2B)AR caused a significant increase in vascular leakage, and agonism of A(2B)AR resulted in marked prolongation of graft survival and suppression of cardiac allograft vasculopathy development. These data suggest a new paradigm in which phosphohydrolysis of adenosine monophosphate by CD73 on graft-resident or circulating cells diminishes transendothelial leukocyte trafficking and mitigates inflammatory and immune sequelae of cardiac transplantation via the A(2B)AR.

Effect of CDT6 on factors of angiogenic balance in tumour cell lines.

BACKGROUND: Cornea-derived transcript 6 (CDT6, also known as AngX) is an angiopoietin-related factor resulting in anti-tumour effect in vivo. However, a recent abstract reported that CDT6 can also induce angiogenesis and promotes tumour growth. In our previous work, CDT6 had failed to show pro- or anti-angiogenic effects. It is unknown if CDT6 expression occurs in human cancer. MATERIALS AND METHODS: An array of human tumour cell lines and tumour tissues was tested for CDT6-gene expression using RT-PCR. To address the controversial role of CDT6 on angiogenesis in different tumour models, the expression levels of four factors of the angiogenic balance (VEGF, endostatin, TIMP-1 and PAI-1) were determined in CDT6-transfected and control cells of the human and murine melanoma cell lines BLM and B16-F10. Endostatin was significantly up-regulated by CDT6 expression in the human model and significantly down-regulated in the mouse model. None of 18 cell lines or 23 tumours expressed CDT6. CONCLUSION: This contradictory effect on endostatin expression in human and mouse models may be an explanation for the conflicting results for the effect of CDT6 expression on angiogenesis.

CDT6-expression can alter tumor sensitivity to chemotherapy.

BACKGROUND: Cornea-derived transcript 6 (CDT6 = AngX) has been shown to have an anti-tumor effect. MATERIALS AND METHODS: We transfected the murine melanoma cell line B16-F10 with the CDT6 gene and compared the sensitivity to cytostatic drugs of the resulting cell line, B16-CDT6, to that of the empty vector-transfected control cell line B16-CMV. RESULTS: The B16-CDT6 line showed a significantly increased sensitivity to doxorubicin as well as a tendency towards a decreased sensitivity to cisplatin. To further resolve the mechanism of the increase in sensitivity to doxorubicin, we also tested the cytotoxic agents vincristine, etoposide and taxol. However no difference in sensitivity for these drugs was found between the B16-CDT6 and the control cell line. CONCLUSION: CDT6 increased the sensitivity to doxorubicin in a mechanism probably not related to interference with the efflux pumps MRP, Pgp or the DNA-associated target enzyme topoisomerase II. Altered gene expression induced by gene therapy might influence tumor sensitivity to chemotherapy.

Effects of the CDT6/ANGX gene on tumour growth in immune competent mice.

BACKGROUND: Cornea-derived transcript 6 (CDT6, also known as AngX) has been described to inhibit tumour growth in a human melanoma growing in nude mice. In another report, however, enhancement of tumour growth in comparable circumstances occurred. We therefore studied the effect of the same gene in different circumstances, using an immune competent mouse model. MATERIALS AND METHODS: In this report we describe the generation of a stably CDT6-expressing clone of the murine melanoma cell line B16-F10. These cells were implanted in female C57/B16 mice with empty vector transfected cells as control. RESULTS: We found no significant inhibition or stimulation of either lag-time or doubling-time of the tumours. In addition no effect of the CDT6 gene product was found on proliferation of endothelial cells in vitro. CONCLUSION: In contrast to an immune deficient mouse model, no anti-tumour effect could be detected with the CDT6 gene product in an immune competent mouse model.

In vivo evaluation of vascular-targeted spheroidal microparticles for imaging and drug delivery application in atherosclerosis.

OBJECTIVE: Vascular-targeting remains a promising strategy for improving the diagnosis and treatment of coronary artery disease (CAD) by providing localized delivery of imaging and therapeutic agents to atherosclerotic lesions. In this work we evaluate how size and shape affects the capacity for a vascular-targeted carrier system to bind inflamed endothelial cells over plaque using ApoE -/- mice with developed atherosclerosis. METHOD: We investigated the adhesion levels along mouse aortae of ellipsoidal and spherical particles targeted to the inflammatory molecules E-selectin and VCAM-1, as well as the biodistribution of targeted and untargeted particles in major organs following injection via tail-vein and a 30-min circulation time. RESULTS: We found that targeted ellipsoidal microparticles adhered to mouse aortae at higher levels than microspheres of similar volume, particularly at segments that contained atherosclerotic plaques. Moreover, both ellipsoidal and spherical nanoparticles displayed the same minimal adhesion levels compared to both types of microparticles evaluated, likely due to poor localization of nanoparticles to the vessel wall in blood flow. We found that microparticles targeted to plaque-associated inflammation were retained at higher levels in the lungs than untargeted particles, largely due to molecular interaction with the pulmonary endothelium. The level of the mechanical entrapment of ellipsoidal microparticles in the lungs was also not significantly different from that of microspheres of the same volume despite a ∼3-fold higher major axis length for the ellipsoids. CONCLUSIONS: Particle shape and size should be considered in the design of carrier systems to target atherosclerosis, as these parameters can be tuned to improve carrier performance.

Endothelial Cx40 limits myocardial ischaemia/reperfusion injury in mice.

AIMS: Gap junctions are indispensable for the function of heart and blood vessels by providing electrical coupling and direct cell-to-cell transfer of small signalling molecules. Gap junction channels between neighbouring cells are composed of 12 connexins (Cx). Changes in Cx43 expression, localization, and channel properties in cardiomyocytes contribute to infarction and reperfusion injury of the heart. It is increasingly recognized that deleterious consequences of ischaemia/reperfusion (IR) are modulated by the inflammatory response and endothelial function. The role of the endothelial connexins, i.e. Cx40 and Cx37, in cardiac IR injury is, however, not known. METHODS AND RESULTS: Following 30 min ischaemia and 24 h reperfusion, we found a significant increase in myocardial infarct size in mice with endothelial-specific deletion of Cx40 (Cx40del), but not in Cx37-deficient mice. The cardioprotective effect of endothelial Cx40 was associated with a decrease in neutrophil infiltration. Moreover, beneficial effects of endothelial Cx40 were not observed in isolated Langendorff-perfused hearts, suggesting direct involvement of endothelial-leucocyte interactions in the cardiac injury. Single-dose administration of methotrexate, a CD73 activator, reduced infarct size and neutrophil infiltration into the infarcted myocardium in Cx40del but not in control mice. Similar to Cx40del mice, CD73-deficient mice showed increased sensitivity to cardiac IR injury, which could not be conversed by methotrexate. CONCLUSION: Endothelial Cx40, but not Cx37, is implicated in resistance of the heart to IR injury by activation of the CD73 pathway. Thus, the Cx40-CD73 axis may represent an interesting target for controlling reperfusion damage associated with revascularization in coronary disease.

Increased CD39 nucleotidase activity on microparticles from patients with idiopathic pulmonary arterial hypertension.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a devastating disease characterized by increased pulmonary vascular resistance, smooth muscle and endothelial cell proliferation, perivascular inflammatory infiltrates, and in situ thrombosis. Circulating intravascular ATP, ADP, AMP and adenosine activate purinergic cell signaling pathways and appear to induce many of the same pathologic processes that underlie IPAH. Extracellular dephosphorylation of ATP to ADP and AMP occurs primarily via CD39 (ENTPD1), an ectonucleotidase found on the surface of leukocytes, platelets, and endothelial cells. Microparticles are micron-sized phospholipid vesicles formed from the membranes of platelets and endothelial cells. OBJECTIVES: Studies here examine whether CD39 is an important microparticle surface nucleotidase, and whether patients with IPAH have altered microparticle-bound CD39 activity that may contribute to the pathophysiology of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Kinetic parameters, inhibitor blocking experiments, and immunogold labeling with electron microscopy support the role of CD39 as a major nucleotidase on the surface of microparticles. Comparison of microparticle surface CD39 expression and nucleotidase activity in 10 patients with advanced IPAH and 10 healthy controls using flow cytometry and thin layer chromatograph demonstrate the following: 1) circulating platelet (CD39(+)CD31(+)CD42b(+)) and endothelial (CD39(+)CD31(+)CD42b(-)) microparticle subpopulations in patients with IPAH show increased CD39 expression; 2) microparticle ATPase and ADPase activity in patients with IPAH is increased. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time increased CD39 expression and function on circulating microparticles in patients with IPAH. Further research is needed to elucidate whether these findings identify an important trigger for the development of the disease, or reflect a physiologic response to IPAH.

Targeting therapeutics to the vascular wall in atherosclerosis--carrier size matters.

OBJECTIVE: Vascular-targeted imaging and drug delivery systems are promising for the treatment of atherosclerosis due to the vast involvement of endothelium in the initiation and growth of plaque. Herein, we investigated the role of particle size in dictating the ability of vascular-targeted spherical particles to interact with the vascular wall (VW) from pulsatile and recirculating human blood flow relevant in atherosclerosis. METHODS: In vitro parallel plate flow chambers (PPFC) with straight or vertical step channel were used to examine the localization and binding efficiency of inflammation-targeted polymeric spheres sized from 0.2 to 5 µm to inflamed endothelium from disturbed reconstituted and whole blood flow. Apolipoprotein deficient mice were used to study particle localization and binding to plaque in vivo. RESULTS: The efficiency of particle binding in disturbed reconstituted blood flow increases as spherical diameter increases from 500 nm to 5 µm. No significant difference was observed between adhesion of 200 nm and 500 nm spheres. Binding efficiency for all particle size was enhanced in disturbed whole blood flow except adhesion of 5 µm in pulsatile whole blood. The adhesion trend in the in vivo model confirmed the binding pattern observed in in vitro assays. CONCLUSIONS: The presented data shows that the binding efficiency of vascular-targeted drug carriers in blood flow is a function of particle size, wall shear rate, flow type, blood composition and ligand characteristics. Overall, the presented results suggest that micron-sized spherical particles (2 µm), not nanospheres, are optimal for vascular-targeted drug delivery applications in medium to large vessel relevant in atherosclerosis.

Self-regulation of inflammatory cell trafficking in mice by the leukocyte surface apyrase CD39.

Leukocyte and platelet accumulation at sites of cerebral ischemia exacerbate cerebral damage. The ectoenzyme CD39 on the plasmalemma of endothelial cells metabolizes ADP to suppress platelet accumulation in the ischemic brain. However, the role of leukocyte surface CD39 in regulating monocyte and neutrophil trafficking in this setting is not known. Here we have demonstrated in mice what we believe to be a novel mechanism by which CD39 on monocytes and neutrophils regulates their own sequestration into ischemic cerebral tissue, by catabolizing nucleotides released by injured cells, thereby inhibiting their chemotaxis, adhesion, and transmigration. Bone marrow reconstitution and provision of an apyrase, an enzyme that hydrolyzes nucleoside tri- and diphosphates, each normalized ischemic leukosequestration and cerebral infarction in CD39-deficient mice. Leukocytes purified from Cd39-/- mice had a markedly diminished capacity to phosphohydrolyze adenine nucleotides and regulate platelet reactivity, suggesting that leukocyte ectoapyrases modulate the ambient vascular nucleotide milieu. Dissipation of ATP by CD39 reduced P2X7 receptor stimulation and thereby suppressed baseline leukocyte alphaMbeta2-integrin expression. As alphaMbeta2-integrin blockade reversed the postischemic, inflammatory phenotype of Cd39-/- mice, these data suggest that phosphohydrolytic activity on the leukocyte surface suppresses cell-cell interactions that would otherwise promote thrombosis or inflammation. These studies indicate that CD39 on both endothelial cells and leukocytes reduces inflammatory cell trafficking and platelet reactivity, with a consequent reduction in tissue injury following cerebral ischemic challenge.

A review on pro- and anti-angiogenic factors as targets of clinical intervention.

Angiogenesis plays an important role in physiology and pathology. It is a tightly regulated process, influenced by the microenvironment and modulated by a multitude of pro- and anti-angiogenic factors. A thorough understanding of the angiogenic process may lead to novel therapies to target ischemic vascular diseases as well as diseases characterised by excessive angiogenesis such as rheumatoid arthritis, psoriasis or tumours. This review gives an overview of the (groups of) factors involved in different steps of the angiogenic process, divided into factors affecting endothelial proliferation and migration and factors affecting blood coagulation, fibrinolysis and the degradation of basement membranes and the extra-cellular matrix, with a specific emphasis on angiopoietins and their related growth factors. The therapeutic implications of these factors are discussed.

combination of vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP) to improve angiogenic gene therapy.

To improve current angiogenic gene therapy with a vascular endothelial growth factor (VEGF)-encoding plasmid (Baumgartner et al. Circulation 1998; 97: 1114-23 [1]; Kusumanto et al. Fifth Annual Meeting of the American Society of Gene Therapy, Boston, 2002, Abstr. 621 [2]), we have generated a combination plasmid, encoding the VEGF gene and the thymidine phosphorylase (TP, also known as platelet-derived endothelial growth factor (PD-ECGF) or gliostatin (GLS)) gene: phVEGF165-TP.MB. Upon transfection in COS-7 cells both gene products were expressed and functional as shown by Western blots, ELISAs and bioassays. Culture supernatants of COS-7 cells transfected with this plasmid were able to induce endothelial proliferation. In an in vitro angiogenesis assay with recombinant proteins, TP was able to increase VEGF-induced tube formation. The phVEGF165-TP.MB plasmid is therefore a promising candidate for in vivo angiogenesis studies.