Investigating the role of autotaxin in pathogenesis of chronic inflammatory diseases

Rheumatoid arthritis (RA) is a common and destructive arthropathy imposing asubstantial socioeconomic burden. RA pathogenesis is characterized by chronicinflammation of the joints eventually leading to the destruction of cartilage and bone.Inflammation is initially localized in the synovial membrane, that becomes markedlythickened due to synovial cell proliferation and infiltration by inflammatory cells.Synovial fibroblasts (SFs) are among the dominant cell types of the arthritic synoviumthat under the influence of the inflammatory milieu and/or possible epigeneticchanges become activated and hyperplastic releasing a number of effector signalsincluding pro-inflammatory factors and tissue remodeling enzymes. Here we showthat activated RA SFs from both human patients and animal models expresssignificant quantities of autotaxin (ATX, ENPP2), originally isolated as an autocrinetumor motility stimulation factor. We show that ATX expression from SFs is inducedby TNF, and that lysophosphatidic acid (LPA), the enzymatic product of ATX, in turninduces SF activation and effector functions. Conditional genetic ablation of ATXfrom SFs or systemic pharmacological inhibition of ATX/LPA results in diseaseattenuation in animal models of RA, thus establishing the ATX/LPA axis as a novelplayer in chronic inflammation and RA pathogenesis, and as a promising therapeutictarget.Η Ρευματοειδής Αρθρίτιδα (ΡΑ) αποτελεί μια συστηματική χρόνια αρθροπάθεια πουχαρακτηρίζεται από παρατεταμένη φλεγμονή του αρθρικού υμένα. Ο αυξημένοςπολλαπλασιασμός των κυττάρων και η διήθηση από φλεγμονώδη κύτταρα οδηγείστην υπερπλασία του αρθρικού υμένα και τελικά στη διάβρωση του χόνδρου και τωνοστών. Οι ινοβλάστες του αρθρικού υμένα αποτελούν βασικό κυτταρικό τύπο τουιστού που στη ΡΑ, υπό την επίδραση του φλεγμονώδους περιβάλλοντος και πιθανώνεπιγενετικών αλλαγών ενεργοποιούνται και απελευθερώνουν προφλεγμονώδειςπαράγοντες και ένζυμα αναδιοργάνωσης του ιστού.Στην παρούσα μελέτη δείχθηκε ότι οι ενεργοποιημένοι ινοβλάστες στην περίπτωσητης ΡΑ, από ασθενείς αλλά και από πειραματικά μοντέλα ποντικών, εκφράζουν σεμεγάλο βαθμό την πρωτείνη autotaxin (ATX, ENPP2), η οποία απομονώθηκε σαναυτοκρινής παράγοντας κινητικότητας καρκινικών κυττάρων. Η έκφραση της ΑΤΧαπό τους ινοβλάστες επάγεται από τον TNF, ενώ το λυσοφωσφατιδικό οξύ (LPA), τοενζυματικό προϊόν της ΑΤΧ επάγει με τη σειρά του σημαντικές ιδιότητες τωνενεργοποιημένων ινοβλαστών. Η ιστοειδική γενετική απαλοιφή της ΑΤΧ από τουςινοβλάστες είχε σαν αποτέλεσμα τη μείωση των συμπτωμάτων της νόσου στουςποντικούς. Επίσης, προστατευμένοι από τη ΡΑ ήταν και οι ποντικοί στους οποίουςπραγματοποιήθηκε συστηματική φαρμακολογική αναστολή της ΑΤΧ και του LPA.Τα ευρήματα αυτά φανερώνουν το ρόλο του άξονα ATX και LPA στη χρόνιαφλεγμονή και στην παθογένεια της ΡΑ, ενώ προτείνουν τα μόρια αυτά σαν ικανούςφαρμακευτικούς στόχους

A role for bronchial epithelial autotaxin in ventilator-induced lung injury

Background The pathophysiology of acute respiratory distress syndrome (ARDS) may eventually result in heterogeneous lung collapse and edema-flooded airways, predisposing the lung to progressive tissue damage known as ventilator-induced lung injury (VILI). Autotaxin (ATX; ENPP2), the enzyme largely responsible for extracellular lysophosphatidic acid (LPA) production, has been suggested to play a pathogenic role in, among others, pulmonary inflammation and fibrosis. Methods C57BL/6 mice were subjected to low and high tidal volume mechanical ventilation using a small animal ventilator: respiratory mechanics were evaluated, and plasma and bronchoalveolar lavage fluid (BALF) samples were obtained. Total protein concentration was determined, and lung histopathology was further performed Results Injurious ventilation resulted in increased BALF levels of ATX. Genetic deletion of ATX from bronchial epithelial cells attenuated VILI-induced pulmonary edema. Conclusion ATX participates in VILI pathogenesis

Highly Selective Endothelin-1 Receptor A Inhibition Prevents Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice

Background: Pulmonary fibrosis is a chronic disease, which progressively leads to respiratory failure and ultimately death. Endothelin-1 (ET-1), a vasoconstrictor secreted by endothelial cells, promotes vasoconstriction by activation of its receptors A and B. Objectives: We addressed the role of highly selective ET-1 receptor A (ETA) inhibition in the pathogenesis of experimental pulmonary fibrosis by bleomycin (BLM). Methods: BLM sulfate (2 U/mL) or saline was intratracheally administered to C57/Bl6 mice (4 groups; n = 5-11/group). Pretreatment with the highly selective ETA receptor inhibitor sitaxentan (15 mg/kg/day) was started 1 day prior to BLM injection and continued for the duration of the experiment. Lung mechanics were assessed prior to sacrifice at days 7, 14, and 21 after BLM, followed by procurement of bronchoalveolar lavage fluid (BALF), blood, and lung tissue samples. Results: Time-dependent effects of BLM exposure included decreased static compliance and increased lung elastance, airspace inflammation and microvascular permeability, histological acute lung injury and fibrosis, and lung collagen deposition. Pretreatment with highly selective ETA receptor inhibitor had no adverse effect on control mice but improved lung mechanics and lung injury score in addition to decreasing BALF pleocytosis, protein content, and collagen deposition in BLM-treated mice. Mortality from BLM reached 40% and occurred primarily during the inflammatory stage of the model but was abrogated by sitaxentan pretreatment. Conclusions: We conclude that in our BLM-induced pulmonary fibrosis model, prophylactic highly selective ETA inhibition improves survival, preserves lung function, attenuates lung injury, and reduces collagen deposition. (c) 2017 S. Karger AG, Base

Autotaxin Has a Negative Role in Systemic Inflammation

The pathogenesis of sepsis involves complex interactions and a systemic inflammatory response leading eventually to multiorgan failure. Autotaxin (ATX, ENPP2) is a secreted glycoprotein largely responsible for the extracellular production of lysophosphatidic acid (LPA), which exerts multiple effects in almost all cell types through its at least six G-protein-coupled LPA receptors (LPARs). Here, we investigated a possible role of the ATX/LPA axis in sepsis in an animal model of endotoxemia as well as in septic patients. Mice with 50% reduced serum ATX levels showed improved survival upon lipopolysaccharide (LPS) stimulation compared to their littermate controls. Similarly, mice bearing the inducible inactivation of ATX and presenting with >70% decreased ATX levels were even more protected against LPS-induced endotoxemia; however, no significant effects were observed upon the chronic and systemic transgenic overexpression of ATX. Moreover, the genetic deletion of LPA receptors 1 and 2 did not significantly affect the severity of the modelled disease, suggesting that alternative receptors may mediate LPA effects upon sepsis. In translation, ATX levels were found to be elevated in the sera of critically ill patients with sepsis in comparison with their baseline levels upon ICU admission. Therefore, the results indicate a role for ATX in LPS-induced sepsis and suggest possible therapeutic benefits of pharmacologically targeting ATX in severe, systemic inflammatory disorders

Inhibition of HMGCoA reductase by simvastatin protects mice from injurious mechanical ventilation

Background: Mortality from severe acute respiratory distress syndrome exceeds 40% and there is no available pharmacologic treatment. Mechanical ventilation contributes to lung dysfunction and mortality by causing ventilator-induced lung injury. We explored the utility of simvastatin in a mouse model of severe ventilator-induced lung injury. Methods: Male C57BL6 mice (n = 7/group) were pretreated with simvastatin or saline and received protective (8 mL/kg) or injurious (25 mL/kg) ventilation for four hours. Three doses of simvastatin (20 mg/kg) or saline were injected intraperitoneally on days -2, -1 and 0 of the experiment. Lung mechanics, (respiratory system elastance, tissue damping and airway resistance), were evaluated by forced oscillation technique, while respiratory system compliance was measured with quasi-static pressure-volume curves. A pathologist blinded to treatment allocation scored hematoxylin-eosin-stained lung sections for the presence of lung injury. Pulmonary endothelial dysfunction was ascertained by bronchoalveolar lavage protein content and lung tissue expression of endothelial junctional protein Vascular Endothelial cadherin by immunoblotting. To assess the inflammatory response in the lung, we determined bronchoalveolar lavage fluid total cell content and neutrophil fraction by microscopy and staining in addition to Matrix-Metalloprotease-9 by ELISA. For the systemic response, we obtained plasma levels of Tumor Necrosis Factor-a, Interleukin-6 and Matrix-Metalloprotease-9 by ELISA. Statistical hypothesis testing was undertaken using one-way analysis of variance and Tukey’s post hoc tests. Results: Ventilation with high tidal volume (HVt) resulted in significantly increased lung elastance by 3-fold and decreased lung compliance by 45% compared to low tidal volume (LVt) but simvastatin abrogated lung mechanical alterations of HVt. Histologic lung injury score increased four-fold by HVt but not in simvastatin-pretreated mice. Lavage pleocytosis and neutrophilia were induced by HVt but were significantly attenuated by simvastatin. Microvascular protein permeability increase 20-fold by injurious ventilation but only 4-fold with simvastatin. There was a 3-fold increase in plasma Tumor Necrosis Factor-a, a 7-fold increase in plasma Interleukin-6 and a 20-fold increase in lavage fluid Matrix-Metalloprotease-9 by HVt but simvastatin reduced these levels to control. Lung tissue vascular endothelial cadherin expression was significantly reduced by injurious ventilation but remained preserved by simvastatin. Conclusion: High-dose simvastatin prevents experimental hyperinflation lung injury by angioprotective and anti-inflammatory effects

A Metabolically-Stabilized Phosphonate Analog of Lysophosphatidic Acid Attenuates Collagen-Induced Arthritis

<div><p>Rheumatoid arthritis (RA) is a destructive arthropathy with systemic manifestations, characterized by chronic synovial inflammation. Under the influence of the pro-inflammatory milieu synovial fibroblasts (SFs), the main effector cells in disease pathogenesis become activated and hyperplastic while releasing a number of signals that include pro-inflammatory factors and tissue remodeling enzymes. Activated RA SFs in mouse or human arthritic joints express significant quantities of autotaxin (ATX), a lysophospholipase D responsible for the majority of lysophosphatidic acid (LPA) production in the serum and inflamed sites. Conditional genetic ablation of ATX from SFs resulted in attenuation of disease symptoms in animal models, an effect attributed to diminished LPA signaling in the synovium, shown to activate SF effector functions. Here we show that administration of 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl-phosphonate (BrP-LPA), a metabolically stabilized analog of LPA and a dual function inhibitor of ATX and pan-antagonist of LPA receptors, attenuates collagen induced arthritis (CIA) development, thus validating the ATX/LPA axis as a novel therapeutic target in RA.</p></div

Autotaxin Activity in Chronic Subdural Hematoma: A Prospective Clinical Study

Autotaxin (ATX) is the ectoenzyme producing the bulk of lysophosphatidic acid (LPA) in circulation. ATX and LPA-mediated signaling (the ATX-LPA axis) play critical roles in the vascular and nervous system development. In adults, this axis contributes to diverse processes, including coagulation, inflammation, fibroproliferation and angiogenesis under physiological and/or pathophysiological conditions. Given evidence implicating several of these processes in chronic subdural hematoma (CSDH) pathogenesis and development, we assessed ATX activity in CSDH patients. Twenty-eight patients were recruited. Blood and hematoma fluid were collected. Enzymatic assays were used to establish serum and hematoma ATX activity. Enzyme-linked immunosorbent assays were used to establish hematoma beta trace (BT) levels, a cerebrospinal fluid (CSF) marker, in a hematoma. ATX activity was nearly three folds higher in hematoma compared to serum (P < 0.001). There was no significant correlation between BT levels and ATX activity in a hematoma. The present results show, for the first time, that ATX is catalytically active in the hematoma fluid of CSDH patients. Moreover, our findings of significantly elevated ATX activity in hematoma compared to serum, implicate the ATX-LPA axis in CSDH pathophysiology. The CSF origin of ATX could not be inferred with the present results. Additional research is warranted to establish the significance of the ATX-LPA axis in CSDH and its potential as a biomarker and/or therapeutic target

Kinetic parameters of <i>in vitro</i> LPC hydrolysis by recombinant ATX and inhibition constants of BrP-LPA.

<p>The presented values are the means (±std) of two independent experiments.</p>*<p><i>Km values for recombinant ATX from R&D</i>.</p

BrP-LPA inhibits ATX activity <i>ex vivo</i>.

<p><b>A.</b> Effect of BrP-LPA on plasma ATX/lysoPLD hydrolysis of exogenous added 50 μΜ LPC (14:0, 16:0, 18:0). <b>B.</b> Correlation of recovered BrP-LPA in whole blood with the added BrP-LPA. <b>C.</b> Inhibition of endogenous ATX/lysoPLD activity in whole blood <i>ex vivo</i> after the addition of increasing BrP-LPA concentrations (0–10 μΜ). ATX activity was measured in the presence of 1 mM LPC with the TOOS assay. <b>D.</b> LPC levels and <b>E.</b> LPA levels measured in whole blood ex vivo in the absence/presence of different BrP-LPA concentrations (0.03–10 μΜ). <b>F.</b> Per cent residual levels of the indicated LPA species in the presence of increasing amounts of BrP-LPA. Solid lines, best fits of averaged data points. * indicates significant (p<0.05), *** indicates significant (p<0.001) decrease relative to control group.</p