Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis

Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)- prostaglandin E2 (PGE2) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE2 administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE2 receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans

Deregulated Lysophosphatidic Acid Metabolism and Signaling in Liver Cancer

Liver cancer is one of the leading causes of death worldwide due to late diagnosis and scarcity of treatment options. The major risk factor for liver cancer is cirrhosis with the underlying causes of cirrhosis being viral infection (hepatitis B or C), metabolic deregulation (Non-alcoholic fatty liver disease (NAFLD) in the presence of obesity and diabetes), alcohol or cholestatic disorders. Lysophosphatidic acid (LPA) is a bioactive phospholipid with numerous effects, most of them compatible with the hallmarks of cancer (proliferation, migration, invasion, survival, evasion of apoptosis, deregulated metabolism, neoangiogenesis, etc.). Autotaxin (ATX) is the enzyme responsible for the bulk of extracellular LPA production, and together with LPA signaling is involved in chronic inflammatory diseases, fibrosis and cancer. This review discusses the most important findings and the mechanisms related to ATX/LPA/LPAR involvement on metabolic, viral and cholestatic liver disorders and their progression to liver cancer in the context of human patients and mouse models. It focuses on the role of ATX/LPA in NAFLD development and its progression to liver cancer as NAFLD has an increasing incidence which is associated with the increasing incidence of liver cancer. Bearing in mind that adipose tissue accounts for the largest amount of LPA production, many studies have implicated LPA in adipose tissue metabolism and inflammation, liver steatosis, insulin resistance, glucose intolerance and lipogenesis. At the same time, LPA and ATX play crucial roles in fibrotic diseases. Given that hepatocellular carcinoma (HCC) is usually developed on the background of liver fibrosis, therapies that both delay the progression of fibrosis and prevent its development to malignancy would be very promising. Therefore, ATX/LPA signaling appears as an attractive therapeutic target as evidenced by the fact that it is involved in both liver fibrosis progression and liver cancer development

Lipid, DNA and protein oxidative stress markers in chronic livel diseases

Reactive oxygen and nitrogen species (ROS/RNS) are involved in liver damage induced by several conditions such as alcohol abuse, viral hepatitis and metabolic disorders. The aim of our study was to determine whether oxidant stress is a feature of early liver disease and to examine the effect of therapy, hepatic necrosis and fibrogenesis to the levels of oxidative stress markers in blood and urine of patients with various forms of chronic liver diseases. The concentrations of isoprostane, aldehydes such as malonaldialdehyde and 4-hydroxynonenal, protein carbonyls, 3-nitrotyrosine, 8-hydroxydeoxygouanosine, glutathione and the activities of myeloperoxidase, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase were estimated. Measurements were performed in 49 patients with Autoimmune Cholestatic liver disease, 36 patients with Autoimmune Hepatitis, 49 patients with Chronic Hepatitis B, 47 patients with Chronic Hepatitis C, 29 patients with Alcoholic Liver Disease and 28 patients with Non Alcoholic Steatohepatitis. The results were compared with those from 75 healthy controls matched for age and sex. To our knowledge, it is the first time in the literature that a significant number of many biomarkers of both oxidants and antioxidants were determined in patients with in autoimmune liver diseases, Chronic Hepatitis B patients and especially in chronic inactive HBsAg carriers. Free radical production was found to be the highest in Alcoholic Liver Disease and the lowest in Chronic Hepatitis B. Oxidant stress is a significant feature of Autoimmune Hepatitis and Autoimmune Cholestatic liver disease. lnactive HBsAg carriers demonstrated free radical production. Βlood glutathione concentration was significantly lower in all groups of patients compared to controls. Furthermore all the oxidative injury products and the most activities of antioxidant enzymes were increased in all groups of patients with elevated hepatic injury markers compared to patients with normal hepatic injury markers. All the activities of the antioxidants enzymes were reduced whereas the majority of oxidative injury products were increased in cirrhotic patients compared to non cirrhotic. Therapy reduced the markers of oxidative stress in normal levels only in the patients with Chronic Hepatitis C. Treatment for Autoimmune Hepatitis was more effective to ameliorate the production of free radicals than treatment with Autoimmune Cholestatic liver disease. Four major findings have emerged from the present study. Firstly, disturbances in antioxidant enzymes activity seem to be involved in the progress of disease to decompensated liver cirrhosis. Secondly, the generation of ROS/RNS was shown to occur from early stage of all chronic liver diseases. Third, therapy ameliorates the production of free radicals. Fourth, we demonstrated a significant correlation between multiple markers of oxidant stress and histologically assessed liver damage in autoimmune and viral liver diseases.Οι δραστικές μορφές οξυγόνου και αζώτου (ΔΜΟ/ΔΜΑ) εμπλέκονται στην ηπατική βλάβη που επάγεται από αρκετές καταστάσεις όπως είναι η κατάχρηση οινοπνεύματος, η ιογενής ηπατίτιδα και μεταβολικές διαταραχές. Ο σκοπός της παρούσης μελέτης ήταν να προσδιοριστεί το κατά πόσο το οξειδωτικό στρες είναι ένα χαρακτηριστικό του αρχικού σταδίου των ηπατικών νοσημάτων και να εξεταστεί η επίδραση της θεραπείας, της ηπατικής νέκρωσης και ινογένεσης στα επίπεδα των δεικτών οξειδωτικού στρες στο αίμα και στα ούρα ασθενών με διάφορα χρόνια νοσήματα του ήπατος. Εκτιμήθηκαν τα επίπεδα των ισοπροστανίων, των αλδεϋδών (4-ύδροξυνονεννάλη και μηλονική διαλδεΰδη), των πρωτεϊνικών καρβονυλίων, της 3-νιτροτυροσίνης, της 8-υδρόξυ-δεοξυγουανοσίνης, της γλουταθειόνης και οι δραστικότητες της μυελοϋπεροξειδάσης, της υπεροξειδάσης της γλουταθειόνης, της αναγωγάσης της γλουταθειόνης, της υπεροξειδικής δισμουτάσης και της καταλάσης. Οι μετρήσεις πραγματοποιήθηκαν σε 36 ασθενείς με Αυτοάνοση Ηπατίτιδα, 49 ασθενείς με Αυτοάνοσα Χολοστατικά Νοσήματα, 49 ασθενείς με Χρόνια Ηπατίτιδα Β, 47 ασθενείς με Χρόνια Ηπατίτιδα C, 29 ασθενείς με Αλκοολική Ηπατοπάθεια και σε 28 ασθενείς με Μη Αλκοολική Στεατοηπατίτιδα. Τα αποτελέσματα συσχετίσθηκαν με 75 υγιείς εθελοντές παρόμοιας ηλικίας και φύλλου. Είναι η πρώτη φορά στη βιβλιογραφία που μετρώνται τόσοι πολλοί δείκτες οξειδωτικών και αντιοξειδωτικών στα αυτοάνοσα νοσήματα του ήπατος, στη χρόνια Ηπατίτιδα Β και ειδικά στους χρόνιους ανενεργούς φορείς της Ηπατίτιδας Β. Η παραγωγή των ελευθέρων ριζών βρέθηκε να είναι υψηλότερη στην Αλκοολική Ηπατοπάθεια και χαμηλότερη στη Χρόνια Ηπατίτιδα Β. Το οξειδωτικό στρες είναι ένα σημαντικό χαρακτηριστικό της Αυτοάνοσης Ηπατίτιδας και των Αυτοάνοσων Χολοστατικών νοσημάτων του ήπατος. Οι χρόνιοι ανενεργοί φορείς της Ηπατίτιδας Β παρουσίαζαν παραγωγή ελευθέρων ριζών. Η συγκέντρωση της γλουταθειόνης ήταν σημαντικά χαμηλότερη σε όλες τις ομάδες των ασθενών συγκριτικά με τους μάρτυρες. Επιπλέον, όλα τα προϊόντα οξειδωτικής βλάβης και οι δραστικότητες των περισσοτέρων αντιοξειδωτικών ενζύμων αυξήθηκαν στους ασθενείς με αυξημένους δείκτες ηπατικής βλάβης σε σχέση με τους ασθενείς με φυσιολογικούς δείκτες ηπατικής βλάβης. Όλες οι δραστικότητες των αντιοξειδωτικών ενζύμων μειώθηκαν ενώ τα περισσότερα προϊόντα οξειδωτικής βλάβης αυξήθηκαν στους κιρρωτικούς σε σχέση με τους μη κιρρωτικούς. Η θεραπεία μείωσε τους δείκτες οξειδωτικού στρες σε φυσιολογικά επίπεδα μόνο στους ασθενείς με Χρόνια Ηπατίτιδα C. Η θεραπεία των ασθενών με Αυτοάνοση Ηπατίτιδα ήταν πιο αποτελεσματική στο να μειώνει την παραγωγή ελευθέρων ριζών από ότι η θεραπεία των ασθενών με Αυτοάνοσα Χολοστατικά Νοσήματα. Τέσσερα κύρια ευρήματα προκύπτουν από την παρούσα μελέτη. Πρώτον, η διαταραχή της λειτουργίας της αντιοξειδωτικής άμυνας εμπλέκεται στην εξέλιξη των νοσημάτων σε κίρρωση. Δεύτερον, η παραγωγή ΔΜΟ/ΔΜΑ συμβαίνει από τα αρχικά στάδια των χρόνιων ηπατικών νοσημάτων. Τρίτον, η θεραπεία μειώνει ως ένα βαθμό τα προϊόντα οξειδωτικής βλάβης και τέταρτον υπάρχει σημαντική συσχέτιση μεταξύ ποικίλων δεικτών οξειδωτικού στρες και της ιστολογικά εκτιμούμενης ηπατικής βλάβης στα αυτοάνοσα και στα ιογενή νοσήματα του ήπατος

Computer Aided Drug Design Approaches for Identification of Novel Autotaxin (ATX) Inhibitors

Autotaxin (ATX) has become an attractive target with a huge pharmacological and pharmacochemical interest in LPA-related diseases and to date many small organic molecules have been explored as potential ATX inhibitors. As a useful aid in the various efforts of identifying novel effective ATX inhibitors, in silico methods can serve as an important and valuable tool. Especially, Virtual Screening (VS) has recently received increased attention due to the large datasets made available, the development of advanced VS techniques and the encouraging fact that VS has contributed to the discovery of several compounds that have either reached the market or entered clinical trials. Different techniques and workflows have been reported in literature with the goal to prioritize possible potent hits. In this review article several deployed virtual screening strategies for the identification of novel potent ATX inhibitors are described

Dysregulation of the Scribble/YAP/β-catenin axis sustains the fibroinflammatory response in a PKHD1-/- mouse model of congenital hepatic fibrosis

: Congenital hepatic fibrosis (CHF), a genetic cholangiopathy characterized by fibropolycystic changes in the biliary tree, is caused by mutations in the PKHD1 gene, leading to defective fibrocystin (FPC), changes in planar cell polarity (PCP) and increased β-catenin-dependent chemokine secretion. In this study, we aimed at understanding the role of Scribble (a protein involved in PCP), Yes-associated protein (YAP), and β-catenin in the regulation of the fibroinflammatory phenotype of FPC-defective cholangiocytes. Immunohistochemistry showed that compared with wild type (WT) mice, in FPC-defective (Pkhd1del4/del4 ) mice nuclear expression of YAP/TAZ in cystic cholangiocytes, significantly increased and correlated with connective tissue growth factor (CTGF) expression and pericystic fibrosis, while Scribble expression on biliary cyst cells was markedly decreased. Cholangiocytes isolated from WT mice showed intense Scribble immunoreactivity at the membrane, but minimal nuclear expression of YAP, which conversely increased, together with CTGF, after small interfering RNA (siRNA) silencing of Scribble. In FPC-defective cholangiocytes, inhibition of YAP nuclear import reduced β-catenin nuclear expression, and CTGF, integrin β6, CXCL1, and CXCL10 mRNA levels, whereas inhibition of β-catenin signaling did not affect nuclear translocation of YAP. Notably, siRNA silencing of Scribble and YAP in WT cholangiocytes mimics the fibroinflammatory changes of FPC-defective cholangiocytes. Conditional deletion of β-catenin in Pkhd1del4/del4  mice reduced cyst growth, inflammation and fibrosis, without affecting YAP nuclear expression. In conclusion, the defective anchor of Scribble to the membrane facilitates the nuclear translocation of YAP and β-catenin with gain of a fibroinflammatory phenotype. The Scribble/YAP/β-catenin axis is a critical factor in the sequence of events linking the genetic defect to fibrocystic trait of cholangiocytes in CHF

The Bulk of Autotaxin Activity Is Dispensable for Adult Mouse Life.

Autotaxin (ATX, Enpp2) is a secreted lysophospholipase D catalysing the production of lysophosphatidic acid, a pleiotropic growth factor-like lysophospholipid. Increased ATX expression has been detected in a number of chronic inflammatory diseases and different types of cancer, while genetic interventions have proven a role for ATX in disease pathogenesis. Therefore, ATX has emerged as a potential drug target and a large number of ATX inhibitors have been developed exhibiting promising therapeutic potential. However, the embryonic lethality of ATX null mice and the ubiquitous expression of ATX and LPA receptors in adult life question the suitability of ATX as a drug target. Here we show that inducible, ubiquitous genetic deletion of ATX in adult mice, as well as long-term potent pharmacologic inhibition, are well tolerated, alleviating potential toxicity concerns of ATX therapeutic targeting

Pathophysiologic implications of innate immunity and autoinflammation in the biliary epithelium

The most studied physiological function of biliary epithelial cells (cholangiocytes) is to regulate bile flow and composition, in particular the hydration and alkalinity of the primary bile secreted by hepatocytes. After almost three decades of studies it is now become clear that cholangiocytes are also involved in epithelial innate immunity, in inflammation, and in the reparative processes in response to liver damage. An increasing number of evidence highlights the ability of cholangiocyte to undergo changes in phenotype and function in response to liver damage. By participating actively to the immune and inflammatory responses, cholangiocytes represent a first defense line against liver injury from different causes. Indeed, cholangiocytes express a number of receptors able to recognize pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), such as Toll-like receptors (TLR), which modulate their pro-inflammatory behavior. Cholangiocytes can be both the targets and the initiators of the inflammatory process. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, both hereditary and acquired, such as cystic fibrosis-related liver disease and sclerosing cholangiti

Autotaxin and Endotoxin-Induced Acute Lung Injury.

Acute Lung Injury (ALI) is a life-threatening, diffuse heterogeneous lung injury characterized by acute onset, pulmonary edema and respiratory failure. Lipopolysaccharide (LPS) is a common cause of both direct and indirect lung injury and when administered to a mouse induces a lung phenotype exhibiting some of the clinical characteristics of human ALI. Here, we report that LPS inhalation in mice results in increased bronchoalveolar lavage fluid (BALF) levels of Autotaxin (ATX, Enpp2), a lysophospholipase D largely responsible for the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) in biological fluids and chronically inflamed sites. In agreement, gradual increases were also detected in BALF LPA levels, following inflammation and pulmonary edema. However, genetic or pharmacologic targeting of ATX had minor effects in ALI severity, suggesting no major involvement of the ATX/LPA axis in acute inflammation. Moreover, systemic, chronic exposure to increased ATX/LPA levels was shown to predispose to and/or to promote acute inflammation and ALI unlike chronic inflammatory pathophysiological situations, further suggesting a differential involvement of the ATX/LPA axis in acute versus chronic pulmonary inflammation

Animal models of cholangiocarcinoma: What they teach us about the human disease

Despite recent advances, pathogenesis of cholangiocarcinoma, a highly lethal cancer, remains enigmatic. Furthermore, treatment options are still limited and often disappointing. For this reason, in the last few years there has been a mounting interest towards the generation of experimental models able to reproduce the main features associated with this aggressive behavior. Toxic and infestation-induced, genetically engineered and cell implantation rodent models have been generated, contributing to a deeper understanding of the complex cell biology of the tumor, sustained by multiple cell interactions and driven by a huge variety of molecular perturbations. Herein, we will overview the most relevant animal models of biliary carcinogenesis, highlighting the methodological strategy, the molecular, histological and clinical phenotypes consistent with the human condition, their particular strengths and weaknesses and the novel therapeutic approaches that have been developed