Ectonucleotidase tri(di)phosphohydrolase-1 (ENTPD-1) disrupts inflammasome/interleukin 1ß-driven venous thrombosis.

Deep vein thrombosis (DVT), caused by alterations in venous homeostasis is the third most common cause of cardiovascular mortality; however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39-deficiency conferred a >2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This was orchestrated by increased phosphorylation of the p65 subunit of NFκB, activation of the NLRP3 inflammasome, and interleukin-1ß (IL-1ß) release in CD39-deficient mice. Substantiating these findings, an IL-1ß-neutralizing antibody attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1ß is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways, and is a critical vascular checkpoint in venous thrombosis.

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.

Ectonucleotidase CD39-driven control of postinfarction myocardial repair and rupture.

Mechanical complications of myocardial infarction (MI) are often fatal. Little is known about endogenous factors that predispose to myocardial rupture after MI. Ectonucleoside triphosphate diphosphohydrolase (CD39) could be a critical mediator of propensity to myocardial rupture after MI due to its role in modulating inflammation and thrombosis. Using a model of permanent coronary artery ligation, rupture was virtually abrogated in cd39-/- mice versus cd39+/+ controls, with elevated fibrin and collagen deposition and marked neutrophil and macrophage influx. Macrophages were found to display increased surface expression of CD301 and CD206, marking a reparative phenotype, driven by increased extracellular ATP and IL-4 in the infarcted myocardium of cd39-/- mice. A myeloid-specific CD39-knockout mouse also demonstrated protection from rupture, with an attenuated rupture phenotype, suggesting that complete ablation of CD39 provides the greatest degree of protection in this model. Absence of CD39, either globally or in a myeloid lineage-restricted fashion, skews the phenotype toward alternatively activated (reparative) macrophage infiltration following MI. These studies reveal a previously unrecognized and unexpected role of endogenous CD39 to skew macrophage phenotype and promote a propensity to myocardial rupture after MI.

Purinergic dysregulation in pulmonary hypertension.

Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.

Suppression of inflammatory cell trafficking and alveolar simplification by the heme oxygenase-1 product carbon monoxide.

Bronchopulmonary dysplasia (BPD), a lung disease of prematurely born infants, is characterized in part by arrested development of pulmonary alveolae. We hypothesized that heme oxygenase (HO-1) and its byproduct carbon monoxide (CO), which are thought to be cytoprotective against redox stress, mitigate lung injury and alveolar simplification in hyperoxia-exposed neonatal mice, a model of BPD. Three-day-old C57BL/6J mice were exposed to air or hyperoxia (FiO2, 75%) in the presence or absence of inhaled CO (250 ppm for 1 h twice daily) for 21 days. Hyperoxic exposure increased mean linear intercept, a measure of alveolar simplification, whereas CO treatment attenuated hypoalveolarization, yielding a normal-appearing lung. Conversely, HO-1-null mice showed exaggerated hyperoxia-induced hypoalveolarization. CO also inhibited hyperoxia-induced pulmonary accumulation of F4/80+, CD11c+, and CD11b+ monocytes and Gr-1+ neutrophils. Furthermore, CO attenuated lung mRNA and protein expression of proinflammatory cytokines, including the monocyte chemoattractant CCL2 in vivo, and decreased hyperoxia-induced type I alveolar epithelial cell CCL2 production in vitro. Hyperoxia-exposed CCL2-null mice, like CO-treated mice, showed attenuated alveolar simplification and lung infiltration of CD11b+ monocytes, consistent with the notion that CO blocks lung epithelial cell cytokine production. We conclude that, in hyperoxia-exposed neonatal mice, inhalation of CO suppresses inflammation and alveolar simplification.

Glycogen synthase kinase-3ß/ß-catenin signaling regulates neonatal lung mesenchymal stromal cell myofibroblastic differentiation.

In bronchopulmonary dysplasia (BPD), alveolar septa are thickened with collagen and α-smooth muscle actin-, transforming growth factor (TGF)-ß-positive myofibroblasts. We examined the biochemical mechanisms underlying myofibroblastic differentiation, focusing on the role of glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin signaling pathway. In the cytoplasm, ß-catenin is phosphorylated on the NH(2) terminus by constitutively active GSK-3ß, favoring its degradation. Upon TGF-ß stimulation, GSK-3ß is phosphorylated and inactivated, allowing ß-catenin to translocate to the nucleus, where it activates transcription of genes involved in myofibroblastic differentiation. We examined the role of ß-catenin in TGF-ß1-induced myofibroblastic differentiation of neonatal lung mesenchymal stromal cells (MSCs) isolated from tracheal aspirates of premature infants with respiratory distress. TGF-ß1 increased ß-catenin expression and nuclear translocation. Transduction of cells with GSK-3ß S9A, a nonphosphorylatable, constitutively active mutant that favors ß-catenin degradation, blocked TGF-ß1-induced myofibroblastic differentiation. Furthermore, transduction of MSCs with ΔN-catenin, a truncation mutant that cannot be phosphorylated on the NH(2) terminus by GSK-3ß and is not degraded, was sufficient for myofibroblastic differentiation. In vivo, hyperoxic exposure of neonatal mice increases expression of ß-catenin in α-smooth muscle actin-positive myofibroblasts. Similar changes were found in lungs of infants with BPD. Finally, low-passage unstimulated MSCs from infants developing BPD showed higher phospho-GSK-3ß, ß-catenin, and α-actin content compared with MSCs from infants not developing this disease, and phospho-GSK-3ß and ß-catenin each correlated with α-actin content. We conclude that phospho-GSK-3ß/ß-catenin signaling regulates α-smooth muscle actin expression, a marker of myofibroblast differentiation, in vitro and in vivo. This pathway appears to be activated in lung mesenchymal cells from patients with BPD.

Neonatal periostin knockout mice are protected from hyperoxia-induced alveolar simplication.

In bronchopulmonary dysplasia (BPD), alveolar septae are thickened with collagen and α-smooth muscle actin, transforming growth factor (TGF)-ß-positive myofibroblasts. Periostin, a secreted extracellular matrix protein, is involved in TGF-ß-mediated fibrosis and myofibroblast differentiation. We hypothesized that periostin expression is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice, an animal model for this disease. We also examined periostin expression in neonatal lung mesenchymal stromal cells and lung tissue of hyperoxia-exposed neonatal mice and human infants with BPD. Two-to-three day-old wild-type and periostin null mice were exposed to air or 75% oxygen for 14 days. Mesenchymal stromal cells were isolated from tracheal aspirates of premature infants. Hyperoxic exposure of neonatal mice increased alveolar wall periostin expression, particularly in areas of interstitial thickening. Periostin co-localized with α-smooth muscle actin, suggesting synthesis by myofibroblasts. A similar pattern was found in lung sections of infants dying of BPD. Unlike wild-type mice, hyperoxia-exposed periostin null mice did not show larger air spaces or α-smooth muscle-positive myofibroblasts. Compared to hyperoxia-exposed wild-type mice, hyperoxia-exposed periostin null mice also showed reduced lung mRNA expression of α-smooth muscle actin, elastin, CXCL1, CXCL2 and CCL4. TGF-ß treatment increased mesenchymal stromal cell periostin expression, and periostin treatment increased TGF-ß-mediated DNA synthesis and myofibroblast differentiation. We conclude that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis.

Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3beta and p70 ribosomal S6 kinase.

Increased medial arterial thickness is a structural change in pulmonary arterial hypertension (PAH). The role of smooth muscle hypertrophy in this process has not been well studied. Bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-beta1, serotonin (or 5-hydroxytryptamine; 5-HT), and endothelin (ET)-1 have been implicated in PAH pathogenesis. We examined the effect of these mediators on human pulmonary artery smooth muscle cell size, contractile protein expression, and contractile function, as well on the roles of glycogen synthase kinase (GSK)-3beta and p70 ribosomal S6 kinase (p70S6K), two proteins involved in translational control, in this process. Unlike epidermal growth factor, BMP-4, TGF-beta1, 5-HT, and ET-1 each increased smooth muscle cell size, contractile protein expression, fractional cell shortening, and GSK-3beta phosphorylation. GSK-3beta inhibition by lithium or SB-216763 increased cell size, protein synthesis, and contractile protein expression. Expression of a non-phosphorylatable GSK-3beta mutant blocked BMP-4-, TGF-beta1-, 5-HT-, and ET-1-induced cell size enlargement, suggesting that GSK-3beta phosphorylation is required and sufficient for cellular hypertrophy. However, BMP-4, TGF-beta1, 5-HT, and ET-1 stimulation was accompanied by an increase in serum response factor transcriptional activation but not eIF2 phosphorylation, suggesting that GSK-3beta-mediated hypertrophy occurs via transcriptional, not translational, control. Finally, BMP-4, TGF-beta1, 5-HT, and ET-1 treatment induced phosphorylation of p70S6K and ribosomal protein S6, and siRNAs against p70S6K and S6 blocked the hypertrophic response. We conclude that mediators implicated in the pathogenesis of PAH induce pulmonary arterial smooth muscle hypertrophy. Identification of the signaling pathways regulating vascular smooth muscle hypertrophy may define new therapeutic targets for PAH.