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

ABSTRACT: 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.

SUBMITTER: Anyanwu AC

PROVIDER: S-EPMC6467508 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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

Anyanwu Anuli C AC Kanthi Yogendra Y Fukase Keigo K Liao Hui H Mimura Tekashi T Desch Karl C KC Gruca Martin M Kaskar Saabir S Sheikh-Aden Hussein H Chi Liguo L Zhao Raymond R Yadav Vinita V Wakefield Thomas W TW Hyman Matthew C MC Pinsky David J DJ

Arteriosclerosis, thrombosis, and vascular biology 20190401 4

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 ...[more]

PMID: 30816804

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Project description:BackgroundAn important mechanism, by which cancer cells achieve immune escape, is the release of extracellular adenosine into their microenvironment. Adenosine activates adenosine A2A and A2B receptors on immune cells constituting one of the strongest immunosuppressive mediators. In addition, extracellular adenosine promotes angiogenesis, tumor cell proliferation, and metastasis. Cancer cells upregulate ectonucleotidases, most importantly CD39 and CD73, which catalyze the hydrolysis of extracellular ATP to AMP (CD39) and further to adenosine (CD73). Inhibition of CD39 is thus expected to be an effective strategy for the (immuno)therapy of cancer. However, suitable small molecule inhibitors for CD39 are not available. Our aim was to identify drug-like CD39 inhibitors and evaluate them in vitro.MethodsWe pursued a repurposing approach by screening a self-compiled collection of approved, mostly ATP-competitive protein kinase inhibitors, on human CD39. The best hit compound was further characterized and evaluated in various orthogonal assays and enzyme preparations, and on human immune and cancer cells.ResultsThe tyrosine kinase inhibitor ceritinib, a potent anticancer drug used for the treatment of anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer, was found to strongly inhibit CD39 showing selectivity versus other ectonucleotidases. The drug displays a non-competitive, allosteric mechanism of CD39 inhibition exhibiting potency in the low micromolar range, which is independent of substrate (ATP) concentration. We could show that ceritinib inhibits ATP dephosphorylation in peripheral blood mononuclear cells in a dose-dependent manner, resulting in a significant increase in ATP concentrations and preventing adenosine formation from ATP. Importantly, ceritinib (1-10 µM) substantially inhibited ATP hydrolysis in triple negative breast cancer and melanoma cells with high native expression of CD39.ConclusionsCD39 inhibition might contribute to the effects of the powerful anticancer drug ceritinib. Ceritinib is a novel CD39 inhibitor with high metabolic stability and optimized physicochemical properties; according to our knowledge, it is the first brain-permeant CD39 inhibitor. Our discovery will provide the basis (i) to develop more potent and balanced dual CD39/ALK inhibitors, and (ii) to optimize the ceritinib scaffold towards interaction with CD39 to obtain potent and selective drug-like CD39 inhibitors for future in vivo studies.

Project description:The pathogenesis of primary sclerosing cholangitis (PSC) and the mechanistic link to inflammatory bowel disease remain ill-defined. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1)/clusters of differentiation (CD) 39, the dominant purinergic ecto-enzyme, modulates intestinal inflammation. Here, we have explored the role of CD39 in biliary injury and fibrosis. The impact of CD39 deletion on disease severity was studied in multidrug resistance protein 2 (Mdr2)-/- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse models of sclerosing cholangitis and biliary fibrosis. Antibody-mediated CD8+ T-cell depletion, selective gut decontamination, experimental colitis, and administration of stable adenosine triphosphate (ATP) agonist were performed. Retinoic acid-induced gut imprinting on T cells was studied in vitro. Over half of Mdr2-/-;CD39-/- double mutants, expected by Mendelian genetics, died in utero. Compared to Mdr2-/-;CD39+/+, surviving Mdr2-/-;CD39-/- mice demonstrated exacerbated liver injury, fibrosis, and ductular reaction. CD39 deficiency led to a selective increase in hepatic CD8+ T cells and integrin α4β7, a T-cell gut-tropism receptor. CD8+ cell depletion in Mdr2-/-;CD39-/- mice diminished hepatobiliary injury and fibrosis. Treatment with antibiotics attenuated, whereas dextran sulfate sodium-induced colitis exacerbated, liver fibrosis in Mdr2-/- mice. Colonic administration of αβ-ATP into CD39-sufficient Mdr2-/- mice triggered hepatic CD8+ cell influx and recapitulated the severe phenotype observed in Mdr2-/-;CD39-/- mice. In vitro, addition of ATP promoted the retinoic acid-induced imprinting of gut-homing integrin α4β7 on naive CD8+ cells. CD39 expression was relatively low in human normal or PSC livers but abundantly present on immune cells of the colon and further up-regulated in samples of patients with inflammatory bowel disease. Conclusion: CD39 deletion promotes biliary injury and fibrosis through gut-imprinted CD8+ T cells. Pharmacological modulation of purinergic signaling may represent a promising approach for the treatment of PSC. (Hepatology Communications 2017;1:957-972).

Dataset Information

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

Publications

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

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