Diane Bouis

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.

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.

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 A2B adenosine receptor (A2BAR), 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 A2BAR caused a significant increase in vascular leakage, and agonism of A2BAR 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 A2BAR.

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.

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.

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 [1]. 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.

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.