Cd39-null Mice Research Articles

Inhibition of Vascular Endothelial Growth Factor Protects against the Development of Oxaliplatin-Induced Sinusoidal Obstruction Syndrome in Wild-Type but Not in CD39-Null Mice

(1) Background: Sinusoidal obstruction syndrome (SOS) after oxaliplatin-based chemotherapy is associated with unfavorable outcomes after partial hepatectomy for colorectal liver metastases (CLM). Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), may prevent SOS development. We investigated the impact of VEGF-inhibition on the development of SOS in a murine model. (2) Methods: Male wild-type and CD39-null mice received oxaliplatin, additional anti-VEGF (OxAV), or controls, and were sacrificed or subjected to major partial hepatectomy (MH). Specimen were used for histological analysis of SOS. Liver damage was assessed by plasma transaminases. The VEGF pathway was elucidated by quantitative PCR of liver tissue and protein analysis of plasma. (3) Results: Mice treated with oxaliplatin developed SOS. Concomitant anti-VEGF facilitated a reduced incidence of SOS, but not in CD39-null mice. SOS was associated with increased plasma VEGF-A and decreased hepatocyte growth factor (HGF). After OxAV treatment, VEGF-R2 was upregulated in wild-type but downregulated in CD39-null mice. Oxaliplatin alone was associated with higher liver damage after MH than in mice with concomitant VEGF-inhibition. (4) Conclusions: We established a murine model of oxaliplatin-induced SOS and provided novel evidence on the protective effect of VEGF-inhibition against the development of SOS that may be associated with changes in the pathway of VEGF and its receptor VEGF-R2.

Endothelial-Targeted CD39 Ameliorates Pulmonary Hypertension in a Murine Model of Endothelial Toxin Administration

Background: Endothelial toxicity (by toxins, viral and bacterial sepsis) and associated inflammation can lead to acute and chronic vasculopathy leading to cardiac compromise. CD39 helps to maintain the endogenous antithrombotic profile of the endothelium. CD39 is an endothelial cell membrane-anchored enzyme that hydrolyses ATP and ADP to AMP. Subsequently, another enzyme CD73 further hydrolyses AMP to adenosine. The consequence of this important pathway is that pro-inflammatory (ATP) and pro-thrombotic (platelet aggregation amplifier, ADP) are converted to adenosine, which is anti-inflammatory, vasodilatory, and anti-thrombotic. CD39 enzyme activity drives this pathway. We have published before that CD39 is anti-thrombotic/anti-inflammatory (N Engl J Med. 2012;367(24):2322-2333; J Clin Invest. 2004;113(10):1440-1446) and also protects from ischaemia-reperfusion injury in heart and kidney (Eur Heart J. 2018;39(2):111-116; Am J Transplant. 2010;10(12):2586-2595) As a human disease correlate, CD39 activity and adenosine signalling is disrupted in human pulmonary hypertension (J Am Heart Assoc. 2020;9:e017404). Moreover, CD39-null mice are predisposed towards the development of experimental pulmonary hypertension after hypoxic injury (Am J Physiol Heart Circ Physiol 311: H286-H298, 2016). In this work, we demonstrate that the administration of a single dose of an endothelial cell toxin (monocrotaline pyrrole) leads to the development of vasculopathy and pulmonary hypertension as characterised by cardiac echo and right ventricular pressure studies. Single administration of endothelial (Vascular cell adhesion molecule, VCAM-1)-targeted soluble CD39 (Anti-VCAM-CD39) ameliorates pulmonary hypertension. Hypothesis: Targeted delivery of CD39 to the pulmonary endothelium will relieve pulmonary hypertension. Methods: 'Anti-VCAM-CD39' is novel therapeutic that contains a scFv recognising VCAM-1 fused to human soluble CD39. It is expressed as a single polypeptide from insect or mammalian cells and affinity purified using a FLAG-affinity tag. Prior work has demonstrated that Anti-VCAM-CD39 administration intravenously leads to dose-dependent prolongation of murine tail bleeding time only at doses above 1mg/kg. We have developed a murine model of pulmonary hypertension by a single administration of monocrotaline pyrrole (MCTP, 8mg/kg). Development of pulmonary hypertension was verified by cardiac echo showing abnormal (decreased) pulmonary acceleration time (PAT): pulmonary ejection (PET) ratio (an accepted parameter of pulmonary hypertension and increased heart weight ratio (heart weight/body weight x100). Pulmonary hypertension was also confirmed by right ventricular systolic pressure (RVSP) measurement. Cardiac echo was done with the Vevo2100 small animal high-frequency ultrasound machine and the 55Mhz MS550s transducer. RVSP was quantified in anaesthetised mice using a Millar pressure catheter (1.4Fr) inserted into the RV, via the right external jugular vein. Results:Development of pulmonary hypertension with single dose of MCTP 8mg/kg shows features of: 1. Increased endothelial VCAM-1 expression (mRNA expression, not shown) 2. Decreased PAT/PET ratios consistent with pulmonary hypertension (Table 1) with typical cardiac echo appearance (Fig. 1) 3. Increased cardiac weight:body weight ratio at D21 (vehicle 0.45±0.05; MCTP 0.6±0.1, p<0.05) 4. Leads to fibrin deposition (Carstairs stain, not shown). Anti-VCAM-CD39 ameliorates pulmonary hypertension, quantitated with cardiac echo and RVSP: 1.Single administration of Anti-VCAM-CD39 at doses of 0.4mg/kg and 1.0mg/kg at 72 h post MCTP improved (increased) PAT/PET ratio at D10 compared with saline control (Fig. 2). 2. Single administration of Anti-VCAM-CD39 at 1.0mg/kg reduced right ventricular systolic pressure (RVSP mmHg) at D10 consistent with amelioration of pulmonary hypertension. (Fig. 3). Effect of lower dose of Anti-VCAM-CD39 at 0.4mg/kg is awaited. Conclusion: 1. MCTP administration cause endothelial activation, VCAM-1 expression and vascular remodelling leading to a model of pulmonary hypertension with right ventricular pressure overload. 2. Single administration of Anti-VCAM-CD39 at doses 0.4mg/kg and 1.0mg/kg (below the threshold prolonging tail-bleeding time) leads to amelioration of pulmonary hypertension suggesting a novel new treatment for this condition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD39 and CD73 Activity Are Protective in a Mouse Model of Antiphospholipid Antibody-Induced Miscarriage

Antiphospholipid antibodies (aPL-ab) including lupus anticoagulant, anticardiolipin (aCL) antibodies and anti-β2-glycoprotein I (β2GP1) antibodies initiate inflammation, tissue factor-activated coagulation, oxidative stress and complement activation. These pathways contribute to the pathophysiology of thrombotic and obstetric manifestations in antiphospholipid syndrome (APS). Not all patients with aPL-ab develop disease phenotype and hence a ‘second hit’ is postulated to be necessary. Extracellular purines (ATP, ADP, and adenosine) are important signalling molecules with the general acceptance that ATP promotes inflammation, ADP is pro platelet-aggregatory, while adenosine has vasodilatory, antithrombotic and anti-inflammatory effects. Extracellular ATP/ADP levels are tightly regulated by CD39 and CD73. CD39 hydrolyses ATP and ADP to AMP, which is then hydrolysed to adenosine by the ecto-5'-nucleotidase CD73.We hypothesised that CD39 and CD73 mitigate both vascular thrombosis as well as inflammatory responses in APS. We developed a murine model of APS-miscarriages by passive infusion of aPL-ab harvested from patients into pregnant mice. Comparison made with infusion of non-immune IgG. We applied this model to wild type (WT), CD39-transgenic mice as well as CD39-null and CD73-null mice. Non-immune IgG administration to WT or CD39-/- C57BL/6 mice did not cause miscarriages, whereas aPL-ab-treated WT mice displayed a mean resorption frequency of 3.67%. In comparison, aPL-ab-treated CD39-/- mice showed a significantly higher mean resorption frequency of 20.1% (p<0.001 vs aPL-ab-treated WT; Fig. 1a). aPL-ab-treated CD73-/- mice also had a significantly increased mean resorption frequency of 10.5% (p<0.05 vs aPL-ab-treated WT).We have previously demonstrated that CD39 over expression in BALB/c transgenic mice is associated with enhanced adenosine generation under prothrombotic conditions. WT BALB/c mice injected with non-immune IgG had a mean resorption frequency of 21.2%, which doubled with aPL-ab injection (39.7%; p<0.05 vs non-immune IgG-treated WT). In transgenic BALB/c mice over-expressing CD39, mean resorption frequencies were significantly lower in the aPL-ab-treated group (11.4% vs 39.7% in aPL-ab-treated WT, p<0.01).QRT-PCR showed 1.6-fold and 2.4-fold increase in TF expression in aPL-ab administered CD39-/- and CD73-/- mice relative to both WT C57BL/6 mice and littermates injected with non-immune sera and. CD39 over expression suppressed TF expression compared with WT BALB/c mice after both non-immune IgG and aPL-ab challenge particularly with 6.3 fold lower TF mRNA levels after aPL-ab infusion. Decidua from aPL-ab injected CD39-/- and CD73-/- C57BL/6 mice showed increased TF immunoreactivity compared to aPL-ab-treated or nonimmune IgG-treated WT mice. C3d deposition was increased by aPL-ab treatment relative to non-immune IgG treatment in all groups except the CD39 transgenic mice (Fig. 3). C3d deposition was most marked in aPL-ab-treated CD39-/- and CD73-/- mice.Placental expression of inflammatory cytokines C57BL/6was measured at the mRNA level by qRT-PCR. TNF-α expression was doubled in CD39-/- and CD73-/- mice relative to non-immune IgG controls. CD39 Tg mice showed suppression of TNF- α after aPL-ab challenge. Consistent with the previously documented role for IL-10 in exacerbating lupus/auto-immune diseases, we noted a significant reduction in IL-10 mRNA in aPL-ab-treated CD39 Tg mice compared to aPL-ab-treated WT mice.Therefore, we demonstrate a protective role for CD39 in APS and provide rationale for the development of soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease DisclosuresDwyer:Kyowa Kirin Pharmaceutical Development, Inc.: Employment.

Tuning the Thromboinflammatory Response to Venous Flow Interruption by the Ectonucleotidase CD39.

Objective- Leukocyte flux contributes to thrombus formation in deep veins under pathological conditions, but mechanisms that inhibit venous thrombosis are incompletely understood. Ectonucleotide di(tri)phosphohydrolase 1 ( ENTPD1 or Cd39), an ectoenzyme that catabolizes extracellular adenine nucleotides, is embedded on the surface of endothelial cells and leukocytes. We hypothesized that under venous stasis conditions, CD39 regulates inflammation at the vein:blood interface in a murine model of deep vein thrombosis. Approach and Results- CD39-null mice developed significantly larger venous thrombi under venous stasis, with more leukocyte recruitment compared with wild-type mice. Gene expression profiling of wild-type and Cd39-null mice revealed 76 differentially expressed inflammatory genes that were significantly upregulated in Cd39-deleted mice after venous thrombosis, and validation experiments confirmed high expression of several key inflammatory mediators. P-selectin, known to have proximal involvement in venous inflammatory and thrombotic events, was upregulated in Cd39-null mice. Inferior vena caval ligation resulted in thrombosis and a corresponding increase in both P-selectin and VWF (von Willebrand Factor) levels which were strikingly higher in mice lacking the Cd39 gene. These mice also manifest an increase in circulating platelet-leukocyte heteroaggregates suggesting heterotypic crosstalk between coagulation and inflammatory systems, which is amplified in the absence of CD39. Conclusions- These data suggest that CD39 mitigates the venous thromboinflammatory response to flow interruption.

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner.

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p < 0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p < 0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p < 0.05) and endothelial apoptosis (p < 0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication-with implications in organ regeneration.

Abstract 75: Ischemic Heart Failure is Exacerbated in CD39-null Mice

Background: CD39 (ectonucleoside triphosphate diphosphohydrolase) is a nucleotidase expressed on endothelial cells, vascular smooth muscles cells, and leukocytes. CD39 plays a key role in vascular homeostasis, hydrolyzing extracellular ATP and ADP. CD39 has been shown to be important in models of ischemic preconditioning and cardiac ischemia reperfusion. However, the effect of CD39 activity on functional recovery of heart after myocardial infarction (MI) has not been evaluated. Hypothesis: Genetic ablation of CD39 expression exacerbates post-myocardial infarction cardiac function and fibrosis. Methods: Wild-type (WT) and CD39-null mice were subjected to coronary artery ligation. Cardiac function and protein evaluation of fibrotic markers was performed at day 28 post-MI. Results: Evaluation at Day 28 post-MI revealed that while mice of both genotypes had similarly reduced ejection fraction and equally compromised contractile function (dP/dtmax), there was a more pronounced negative effect on lusitropy (dP/dtmin) and increased left ventricular end-diastolic pressure in CD39-null mice. Therefore, cd39 gene ablation associates with the development of worsening cardiac performance. Histological analysis revealed increased collagen deposition and abundance of alpha-smooth muscle actin (αSMA) positive interstitial cells in the CD39-null hearts compared to WT hearts. To quantify these findings immunoblot analysis for collagen and αSMA was performed. We found that collagen and αSMA were increased at Day 28 post-MI, in CD39-null hearts compared to WT hearts. Conclusion: CD39 ablation has detrimental effects on post-MI recovery, resulting in diminished cardiac performance and increased fibrosis.

Pulmonary Natural Killer T Cells Play an Essential Role in Mediating Hyperoxic Acute Lung Injury

Critically ill patients are routinely exposed to high concentrations of supplemental oxygen for prolonged periods of time, which can be life-saving in the short term, but such exposure also causes severe lung injury and increases mortality. To address this therapeutic dilemma, we studied the mechanisms of the tissue-damaging effects of oxygen in mice. We show that pulmonary invariant natural killer T (iNKT) cells are unexpectedly crucial in the development of acute oxygen-induced lung injury. iNKT cells express high concentrations of the ectonucleotidase CD39, which regulates their state of activation. Both iNKT cell-deficient (Jα18(-/-)) and CD39-null mice tolerate hyperoxia, compared with wild-type control mice that exhibit severe lung injury. An adoptive transfer of wild-type iNKT cells into Jα18(-/-) mice results in hyperoxic lung injury, whereas the transfer of CD39-null iNKT cells does not. Pulmonary iNKT cell activation and proliferation are modulated by ATP-dependent purinergic signaling responses. Hyperoxic lung injury can be induced by selective P2X7-receptor blockade in CD39-null mice. Our data indicate that iNKT cells are involved in the pathogenesis of hyperoxic lung injury, and that tissue protection can be mediated through ATP-induced P2X7 receptor signaling, resulting in iNKT cell death. In conclusion, our data suggest that iNKT cells and purinergic signaling should be evaluated as potential novel therapeutic targets to prevent hyperoxic lung injury.

Increased Plasma Levels of Microparticles Expressing CD39 and CD133 in Acute Liver Injury

We have previously demonstrated that CD133 and CD39 are expressed by hematopoietic stem cells (HSC), which are mobilized after liver injury and target sites of injury, limit vascular inflammation, and boost hepatic regeneration. Plasma microparticles (MP) expressing CD39 can block endothelial activation. Here, we tested whether CD133 MP might be shed in a CD39-dependent manner in a model of liver injury and could potentially serve as biomarkers of liver failure in the clinic. Wild-type and Cd39-null mice were subjected to acetaminophen-induced liver injury. Mice were sacrificed and plasma MP were isolated by ultracentrifugation. HSC and CD133 MP levels were analyzed by fluorescence-activated cell sorting. Patients were enrolled with acute (n=5) and acute on chronic (n=5) liver injury with matched controls (n=7). Blood was collected at admission and plasma CD133 and CD39 MP subsets were analyzed by fluorescence-activated cell sorting. HSC and CD133 MP levels were significantly increased only in the plasma of wild-type mice with acetaminophen hepatotoxicity (P<0.05). No increases in CD133 MP were noted in Cd39-null mice. Plasma MP increases were observed in patients with liver injury. These MP were characterized by significantly higher levels of CD39 (P<0.05). HSC and plasma CD133 MP levels increase in a CD39-dependent manner during experimental acute liver injury. Increased levels of CD39 MP are differentially noted in patients with liver injury. Further research is needed to determine whether MP fluxes are secondary to pathophysiologic insults to the liver or might reflect compensatory responses.

Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase‐1 and NTPDase1/CD39 activities

Extracellular ATP and ADP trigger inflammatory, vasodilatatory, and prothrombotic signaling events in the vasculature, and their turnover is governed by networks of membrane-associated enzymes. The contribution of soluble activities to intravascular nucleotide homeostasis remains controversial. By using thin-layer chromatographic assays, we revealed transphosphorylation of [γ-(32)P]ATP and AMP by human and murine sera, which was progressively inhibited by specific adenylate kinase (AK) inhibitor Ap(5)A. This phosphotransfer reaction was diminished markedly in serum from knockout mice lacking the major AK isoform, AK1, and in human serum immunodepleted of AK1. We also showed that ∼75% ADP in cell-free serum is metabolized via reversible AK1 reaction 2ADP ↔ ATP + AMP. The generated ATP and AMP are then metabolized through the coupled nucleotide pyrophosphatase/phosphodiesterase and 5'-nucleotidase/CD73 reactions, respectively. Constitutive presence of another nucleotide-converting enzyme, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, known as CD39), was ascertained by the relative deficiency of serum from CD39-null mice to dephosphorylate [(3)H]ADP and [γ-(32)P]ATP, and also by diminished [(3)H]ADP hydrolysis by human serum pretreated with NTPDase1 inhibitors, POM-1 and ARL-67156. In summary, we have identified hitherto unrecognized soluble forms of AK1 and NTPDase1/CD39 that contribute in the active cycling between the principal platelet-recruiting agent ADP and other circulating nucleotides.

Liver damage and systemic inflammatory responses are exacerbated by the genetic deletion of CD39 in total hepatic ischemia

Liver ischemia reperfusion injury is associated with both local damage to the hepatic vasculature and systemic inflammatory responses. CD39 is the dominant vascular endothelial cell ectonucleotidase and rapidly hydrolyses both adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate. These biochemical properties, in tandem with 5'-nucleotidases, generate adenosine and potentially illicit inflammatory vascular responses and thrombosis. We have evaluated the role of CD39 in total hepatic ischemia reperfusion injury (IRI). Wildtype mice, Cd39-hemizygous mice (+/-) and matched Cd39-null mice (-/-); (n = 25 per group) underwent 45min of total warm ischemia with full inflow occlusion necessitating partial hepatectomy. Soluble nucleoside triphosphate diphosphohydrolase (NTPDases) or adenosine/amrinone were administered to wildtype (n = 6) and Cd39-null mice (n = 6) in order to study protective effects in vivo. Parameters of liver injury, systemic inflammation, hepatic ATP determinations by P(31)-NMR and parameters of lung injury were obtained. All wildtype mice survived up to 7days with minimal biochemical disturbances and minor evidence for injury. In contrast, 64% of Cd39+/- and 84% of Cd39-null mice required euthanasia or died within 4h post-reperfusion with liver damage and systemic inflammation associated with hypercytokinemia. Hepatic ATP depletion was pronounced in Cd39-null mice posthepatic IRI. Soluble NTPDase or adenosine administration protected Cd39-deficient mice from acute reperfusion injury. We conclude that CD39 is protective in hepatic IRI preventing local injury and systemic inflammation in an adenosine dependent manner. Our data indicate that vascular CD39 expression has an essential protective role in hepatic IRI.

Vascular CD39/ENTPD1 Directly Promotes Tumor Cell Growth by Scavenging Extracellular Adenosine Triphosphate

Extracellular adenosine triphosphate (ATP) is known to boost immune responses in the tumor microenvironment but might also contribute directly to cancer cell death. CD39/ENTPD1 is the dominant ectonucleotidase expressed by endothelial cells and regulatory T cells and catalyzes the sequential hydrolysis of ATP to AMP that is further degraded to adenosine by CD73/ecto-5'-nucleotidase. We have previously shown that deletion of Cd39 results in decreased growth of transplanted tumors in mice, as a result of both defective angiogenesis and heightened innate immune responses (secondary to loss of adenosinergic immune suppression). Whether alterations in local extracellular ATP and adenosine levels as a result of CD39 bioactivity directly affect tumor growth and cytotoxicity has not been investigated to date. We show here that extracellular ATP exerts antitumor activity by directly inhibiting cell proliferation and promoting cancer cell death. ATP-induced antiproliferative effects and cell death are, in large part, mediated through P2X(7) receptor signaling. Tumors in Cd39 null mice exhibit increased necrosis in association with P2X(7) expression. We further demonstrate that exogenous soluble NTPDase, or CD39 expression by cocultured liver sinusoidal endothelial cells, stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP. Collectively, our findings indicate that local expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy in cancer management.

Transgenic Overexpression of CD39 Protects Against Renal Ischemia-Reperfusion and Transplant Vascular Injury

The vascular ectonucleotidases CD39[ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1), EC 3.6.1.5] and CD73[EC 3.1.3.5] generate adenosine from extracellular nucleotides. CD39 activity is critical in determining the response to ischemia-reperfusion injury (IRI), and CD39 null mice exhibit heightened sensitivity to renal IRI. Adenosine has multiple mechanisms of action in the vasculature including direct endothelial protection, antiinflammatory and antithrombotic effects and is protective in several models of IRI. Mice transgenic for human CD39 (hCD39) have increased capacity to generate adenosine. We therefore hypothesized that hCD39 transgenic mice would be protected from renal IRI. The overexpression of hCD39 conferred protection in a model of warm renal IRI, with reduced histological injury, less apoptosis and preserved serum creatinine and urea levels. Benefit was abrogated by pretreatment with an adenosine A2A receptor antagonist. Adoptive transfer experiments showed that expression of hCD39 on either the vasculature or circulating cells mitigated IRI. Furthermore, hCD39 transgenic kidneys transplanted into syngeneic recipients after prolonged cold storage performed significantly better and exhibited less histological injury than wild-type control grafts. Thus, systemic or local strategies to promote adenosine generation and signaling may have beneficial effects on warm and cold renal IRI, with implications for therapeutic application in clinical renal transplantation.

Variable Impact of CD39 in Experimental Murine Colitis

Dysregulation of immune responses in inflammatory bowel diseases (IBD) results in intestinal inflammation and vascular injury while exacerbating systemic disease. CD39 is an ectonucleotidase, expressed by T regulatory cells and dendritic cells, that hydrolyzes extracellular nucleotides to modify those cellular immune responses implicated in IBD. Genetic polymorphisms of CD39 have been linked to Crohn's disease while gene deletion in mice exacerbates dextran sodium sulphate-induced colitis. The aim of this study was to test how global deletion of CD39 in mice impacts other models of experimental colitis. Colitis was induced in CD39-null and -wt mice, using trinitrobenzene sulfonic acid (TNBS, 125 mg/kg) administered intrarectally. Oxazolone colitis (1.5% oxazolone in 50% alcohol) was induced in comparable groups. Morphology, clinical and molecular parameters, and FACS analyses of lamina propria mononuclear cells (LPMC) were examined in CD39-null mice. CD39 expression was analyzed in human IBD biopsies. Paradoxically, TNBS colitis in CD39-null mice was characterized by improved survival, favorable clinical scores, and decreased MPO activity, when compared to wt mice (P < 0.05). LPMC from TNBS colitis contained significantly increased amounts of T-cells (CD3(+) and CD4(+)) and TNF-α mRNA expression were increased over those in CD39 null mice (P < 0.05). In contrast, oxazolone treated CD39-null and wt mice had comparable outcomes. In both ulcerative colitis and Crohn's disease, CD39 is present at high levels in intestinal tissue biopsies. TNBS colitis was attenuated in CD39-null mice whereas oxazolone-induced colitis was not impacted. Impaired adaptive cellular immune reactivity in the CD39-null environment appears protective in hapten-mediated Th1-type colitis. CD39 is expressed at high levels in clinical IBD tissues.

Deletion of Cd39 on Natural Killer Cells Attenuates Hepatic Ischemia/Reperfusion Injury in Mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Vascular stasis, intestinal hemorrhage, and heightened vascular permeability complicate acute portal hypertension in cd39-null mice

Vasoactive factors that regulate splanchnic hemodynamics include nitric oxide, catecholamines, and possibly extracellular nucleosides/nucleotides (adenosine, ATP). CD39/ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1) is the major vascular ectonucleotidase that hydrolyzes extracellular nucleotides. CD39 activity may be modulated by vascular injury, inflammation, and altered oxygen tension. Altered Cd39 expression by the murine hepatosplanchnic vasculature may impact hemodynamics and portal hypertension (PHT) in vivo. We noted that basal portal pressures (PPs) were comparable in wild-type and Cd39-null mice (n = 9). ATP infusions resulted in increments in PP in wild-type mice, but, in contrast, this significantly decreased in Cd39-null mice (n = 9) post-ATP in a nitric oxide-dependent manner. We then studied Cd39/NTPDase1 deletion in the regulation of portal hemodynamics, vascular integrity, and intestinal permeability in a murine model of PHT. Partial portal vein ligation (PPVL) was performed in Cd39-null (n = 44) and wild-type (n = 23) mice. Sequential measurements obtained after PPVL were indicative of comparable levels of PHT (ranges 14-29 mmHg) in both groups. There was one death in the wild-type group and eight in the Cd39-null group from intestinal bleeding (P = 0.024). Circulatory stasis in the absence of overt portal vein thrombosis, portal congestion, intestinal hemorrhage, and increased permeability were evident in all surviving Cd39-null mice. Deletion of Cd39 results in deleterious outcomes post-PPVL that are associated with significant microcirculatory derangements and major intestinal congestion with hemorrhage mimicking acute mesenteric occlusion. Absent Cd39/NTPDase1 and decreased generation of adenosine in the splanchnic circulation cause heightened vascular permeability and gastrointestinal hemorrhage in PPVL.

Preservation of cochlear function in Cd39 deficient mice

Signalling actions of extracellular nucleotides via P2 receptors influence cellular function in most tissues. In the inner ear, P2 receptor signaling is involved in many processes including the regulation of hearing sensitivity and the cochlea’s response to noise stress. CD39 (NTPDase1/ENTPD1) is an ectonucleotidase (ecto-nucleoside triphosphate diphosphohydrolase) that can hydrolyse purine and pyrimidine nucleoside tri- and di-phosphates to generate monophosphate nucleosides. Mice null for Cd39 exhibit major alterations in haemostasis and profound alterations in inflammatory and thrombotic reactions. Studies in the cochlea have suggested the involvement of purinergic-type signals that could be modulated by CD39 in regulation of cochlear blood flow and also auditory neurotransmission. This study aimed to determine the auditory phenotype of adult Cd39 null mice on the C57BL6 background . Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were unaffected in Cd39-deficient mice across the range of test frequencies, suggesting normal neural and outer hair cell function. Mutant mice also showed little difference to wild type mice in vulnerability to acoustic trauma. Gene expression analysis of other membrane-bound NTPDases with comparable hydrolytic activity demonstrated an up-regulation of Entpd2 and Entpd8 in the cochleae of Cd39 deficient mice. These findings suggest that Cd39 deletion alone does not adversely affect cochlear function, possibly as compensatory up-regulation of other surface located NTPDases may offset predicted alterations in cochlear homeostasis.

Vascular smooth muscle cell expression of ectonucleotidase CD39 (ENTPD1) is required for neointimal formation in mice

Vascular smooth muscle cell (VSMC) migration and proliferation are critical steps in the pathogenesis of atherosclerosis, post-angioplasty restenosis, neointimal hyperplasia, and chronic allograft rejection. Extracellular nucleotides are known to influence both migration and proliferation of VSMC. Although it is well established that vascular endothelial Cd39/ENTPD1 regulates blood nucleotide concentrations, whether Cd39 associated with VSMC also impacts vascular wall pathology has not been investigated. The objective of this paper is to determine levels of expression of Cd39 on VSMC and functional consequences of gene deletion in vitro and in vivo. Cd39 is the major ectonucleotidase in VSMC, as shown by substantive decreases in ecto-ATPase and -ADPase activity in Cd39-null cells compared to wild type. Significant decreases in neointimal lesion formation are observed in Cd39-null mice at 21 days post arterial balloon injury. Stimulated Cd39-null VSMC have pronounced proliferative responses in vitro. However, using Transwell systems, we show that Cd39-null VSMC fail to migrate in response to ATP, UTP, and PDGF. Cd39 is the dominant ectonucleotidase expressed by VSMC. Deletion of Cd39 in mice results in decreased neointimal formation after vascular injury and is associated with impaired VSMC migration responses in vitro.

Natural killer T cell dysfunction in CD39-null mice protects against concanavalin A–induced hepatitis

Concanavalin A (Con A)-induced injury is an established natural killer T (NKT) cell-mediated model of inflammation that has been used in studies of immune liver disease. Extracellular nucleotides, such as adenosine triphosphate, are released by Con A-stimulated cells and bind to specific purinergic type 2 receptors to modulate immune activation responses. Levels of extracellular nucleotides are in turn closely regulated by ectonucleotidases, such as CD39/NTPDase1. Effects of extracellular nucleotides and CD39 on NKT cell activation and upon hepatic inflammation have been largely unexplored to date. Here, we show that NKT cells express both CD39 and CD73/ecto-5'-nucleotidase and can therefore generate adenosine from extracellular nucleotides, whereas natural killer cells do not express CD73. In vivo, mice null for CD39 are protected from Con A-induced liver injury and show substantively lower serum levels of interleukin-4 and interferon-gamma when compared with matched wild-type mice. Numbers of hepatic NKT cells are significantly decreased in CD39 null mice after Con A administration. Hepatic NKT cells express most P2X and P2Y receptors; exceptions include P2X3 and P2Y11. Heightened levels of apoptosis of CD39 null NKT cells in vivo and in vitro appear to be driven by unimpeded activation of the P2X7 receptor. CD39 and CD73 are novel phenotypic markers of NKT cells. In turn, CD39 expression [corrected] modulates nucleotide-mediated cytokine production by, and limits apoptosis of, hepatic NKT cells. Deletion of CD39 is protective in [corrected] Con A-induced hepatitis. This study illustrates a [corrected] role for purinergic signaling in NKT-mediated mechanisms that result in liver immune injury.

Disordered Pancreatic Inflammatory Responses and Inhibition of Fibrosis in CD39-Null Mice

Extracellular nucleotides are released from injured cells and bind purinergic-type 2 receptors (P2-Rs) that modulate inflammatory responses. Ectonucleotidases, such as CD39/nucleoside triphosphate diphosphohydrolase-1, hydrolyze extracellular nucleotides to integrate purinergic signaling responses. Because the role of extracellular nucleotides and CD39 in mediating inflammation and fibrosis are understood poorly, we studied the impact of CD39 gene deletion in a model of pancreatic disease. Pancreatitis was induced by cyclosporine pretreatment, followed by cerulein injections (50 mug/kg, 6 intraperitoneal injections/day, 3 times/wk); mice were killed at day 2, week 3, and week 6. Experimental parameters were correlated with cytokine levels in blood, RNA, and protein expression of purinergic and fibrosis markers in tissues. Immunohistochemistry and pancreatic morphometry of fibrosis were performed in wild-type and CD39-null mice. Effects of CD39 deletion on proliferation of primary pancreatic stellate cells (PSCs) were investigated in vitro. Wild-type mice developed morphologic features of pancreatitis with the anticipated development of parenchymal atrophy and fibrosis. CD39 and P2-R became overexpressed in vascular and adventitious wild-type tissues. In contrast, CD39-null mice had inflammatory reactions but developed only minor pancreatic atrophy and limited fibrosis. Interferon-gamma became significantly increased in tissues and plasma of CD39-null mice. Wild-type PSCs expressed high levels of CD39 and P2-R. CD39-null PSCs showed decreased rates of proliferation and the expression of procollagen-alpha1 was inhibited significantly in vitro (P < .03). CD39 deletion decreases fibrogenesis in experimental pancreatitis. Our data implicate extracellular nucleotides as modulators of PSC proliferation and collagen production in pancreatitis.

Abstract 1045: E-ntpdase-1/Cd39 Confers Anti-inflammatory And Immunomodulatory Properties To Circulating Membrane-derived Microparticles

Background: Microparticles (MPs, &lt;1.5ÂμM) are circulating, membrane-derived micro-vesicles shed from cells and platelets under physiological conditions as well as following stress, such as post-myocardial infarction. MPs have been implicated in inflammation, coagulation and vascular function. CD39, the prototype nucleoside triphosphate diphosphohydrolase (NTP-Dase1) within the vasculature modulates extracellular purinergic signalling through ATP and ADP hydrolysis, conferring anti-inflammatory, anti-thrombotic properties to the intravascular space. The aim of this study was to determine if CD39 is present on MPs and whether this impacts MP phenotype and function. Methods: The proteome, absolute numbers as well as cellular origin of circulating MPs from wild type (wt) and cd39-null C57Bl6 mice were compared by 2D-gel electrophoresis, western blot and flow cytometry. Isolated MPs were incubated with endothelial cells (EC) to analyze their intrinsic pro-inflammatory potential. Results: CD39 can be demonstrated and has ecto-enzymatic activity on circulating MPs from wt mice. 2D-gel analysis revealed that cd39-null MPs express lower levels of talin (component of focal adhesion) and ATP synthase, when compared to wild type MPs. Mutant mice have increased numbers of circulating MPs, with significantly higher fractions derived from platelets and endothelium as compared to wt mice (p&lt;0.05). The presence of CD39 on wt MPs significantly attenuates inflammatory responses following incubation with EC as compared to cd39-null MPs (reduced IL-6 and TNF-alpha secretion, ICAM-1 and VCAM-1 expression, vWF release and NF-kappa B activation, p&lt;0.05). Conclusions: CD39 confers anti-inflammatory and immunomodulatory properties upon circulating MPs. These results highlight the protective potential of CD39; a finding that may be exploited therapeutically, with the prospective of administering soluble CD39 (apyrase) to the site of intravascular injury to ameliorate inflammation, as occurs for instance in acute coronary syndromes.