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Background & Aim: The evaluation of liver fibrosis in patients with chronic hepatitis C virus (HCV) infection is important as it is a risk factor for hepatocellular carcinoma. In the recent years, autotaxin (ATX) has been established as a new non-invasive biomarker to predict liver fibrosis. However, antiviral treatment has been reported to decrease serum ATX, but it is unclear whether post-treatment ATX levels reflect liver fibrosis. In the present study, we analyzed the correlation between ATX and liver fibrosis in pre- and post-treatment patients with HCV infection. / Methods: We used 199 samples from 136 patients with HCV infection who had undergone hepatic biopsy before and/or after antiviral treatment at Osaka City University Hospital....

Background. Studies have shown that lipoprotein(a) [Lp(a)], an important carrier of oxidized phospholipids, is causally related to calcific aortic valve stenosis (CAVS). Recently, we found that Lp(a) mediates the development of CAVS through autotaxin (ATX). Objective. To determine the predictive value of circulating ATX mass and activity for CAVS. Methods. We performed a case-control study in 300 patients with coronary artery disease (CAD). Patients with CAVS plus CAD (cases, n = 150) were age- and gender-matched (1 : 1) to patients with CAD without aortic valve disease (controls, n = 150). ATX mass and enzymatic activity and levels of Lp(a) and oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB) were determined in fasting plasma samples. Results. Compared to patients with CAD alone, ATX mass (P < 0.0001), ATX activity (P = 0.05), Lp(a) (P = 0.003) and OxPL-apoB (P < 0.0001) levels were elevated in those with CAVS. After adjustment, we found that ATX mass (OR 1.06, 95% CI 1.03–1.10 per 10 ng mL 1 , P = 0.001) and ATX activity (OR 1.57, 95% CI 1.14–2.17 per 10 RFU min 1 , P = 0.005) were independently associated with CAVS. ATX activity interacted with Lp(a) (P = 0.004) and OxPL-apoB (P = 0.001) on CAVS risk. After adjustment, compared to patients with low ATX activity (dichotomized at the median value) and low Lp(a) (<50 mg dL 1 ) or OxPL-apoB (<2.02 nmol L 1 , median) levels (referent), patients with both higher ATX activity (≥84 RFU min 1 ) and Lp(a) (≥50 mg dL 1 ) (OR 3.46, 95% CI 1.40–8.58, P = 0.007) or OxPL-apoB (≥2.02 nmol L 1 , median) (OR 5.48, 95% CI 2.45–12.27, P < 0.0001) had an elevated risk of CAVS. Conclusion. Autotaxin is a novel and independent predictor of CAVS in patients with CAD.

Background Studies have shown that lipoprotein(a) [Lp(a)], an important carrier of oxidized phospholipids, is causally related to calcific aortic valve stenosis (CAVS). Recently, we found that Lp(a) mediates the development of CAVS through autotaxin (ATX). Objective To determine the predictive value of circulating ATX mass and activity for CAVS. Methods We performed a case-control study in 300 patients with coronary artery disease (CAD). Patients with CAVS plus CAD (cases, n = 150) were age- and gender-matched (1 : 1) to patients with CAD without aortic valve disease (controls, n = 150). ATX mass and enzymatic activity and levels of Lp(a) and oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB) were determined in fasting plasma samples. Results Compared to patients with CAD alone, ATX mass (P < 0.0001), ATX activity (P = 0.05), Lp(a) (P = 0.003) and OxPL-apoB (P < 0.0001) levels were elevated in those with CAVS. After adjustment, we found that ATX mass (OR 1.06, 95% CI 1.03–1.10 per 10 ng mL−1, P = 0.001) and ATX activity (OR 1.57, 95% CI 1.14–2.17 per 10 RFU min−1, P = 0.005) were independently associated with CAVS. ATX activity interacted with Lp(a) (P = 0.004) and OxPL-apoB (P = 0.001) on CAVS risk. After adjustment, compared to patients with low ATX activity (dichotomized at the median value) and low Lp(a) (<50 mg dL−1) or OxPL-apoB (<2.02 nmol L−1, median) levels (referent), patients with both higher ATX activity (≥84 RFU min−1) and Lp(a) (≥50 mg dL−1) (OR 3.46, 95% CI 1.40–8.58, P = 0.007) or OxPL-apoB (≥2.02 nmol L−1, median) (OR 5.48, 95% CI 2.45–12.27, P < 0.0001) had an elevated risk of CAVS. Conclusion Autotaxin is a novel and independent predictor of CAVS in patients with CAD.

Previously considered a degenerative process, cardiovascular calcification is now established as an active process that is regulated in several ways by lipids, phospholipids, and lipoproteins. These compounds serve many of the same functions in vascular and valvular calcification as they do in skeletal bone calcification. Hyperlipidemia leads to accumulation of lipoproteins in the subendothelial space of cardiovascular tissues, which leads to formation of mildly oxidized phospholipids, which are known bioactive factors in vascular cell calcification. One lipoprotein of particular interest is Lp(a), which showed genome-wide significance for the presence of aortic valve calcification and stenosis. It carries an important enzyme, autotaxin, which produces lysophosphatidic acid (LPA), and thus has a key role in inflammation among other functions. Matrix vesicles, extruded from the plasma membrane of cells, are the sites of initiation of mineral formation. Phosphatidylserine, a phospholipid in the membranes of matrix vesicles, is believed to complex with calcium and phosphate ions, creating a nidus for hydroxyapatite crystal formation in cardiovascular as well as in skeletal bone mineralization. This review focuses on the contributions of lipids, phospholipids, lipoproteins, and autotaxin in cardiovascular calcification, and discusses possible therapeutic targets.

Previously considered a degenerative process, cardiovascular calcification is now established as an active process that is regulated in several ways by lipids, phospholipids, and lipoproteins. These compounds serve many of the same functions in vascular and valvular calcification as they do in skeletal bone calcification. Hyperlipidemia leads to accumulation of lipoproteins in the subendothelial space of cardiovascular tissues, which leads to formation of mildly oxidized phospholipids, which are known bioactive factors in vascular cell calcification. One lipoprotein of particular interest is Lp(a), which showed genome-wide significance for the presence of aortic valve calcification and stenosis. It carries an important enzyme, autotaxin, which produces lysophosphatidic acid (LPA), and thus has a key role in inflammation among other functions. Matrix vesicles, extruded from the plasma membrane of cells, are the sites of initiation of mineral formation. Phosphatidylserine, a phospholipid in the membranes of matrix vesicles, is believed to complex with calcium and phosphate ions, creating a nidus for hydroxyapatite crystal formation in cardiovascular as well as in skeletal bone mineralization. This review focuses on the contributions of lipids, phospholipids, lipoproteins, and autotaxin in cardiovascular calcification, and discusses possible therapeutic targets.

Le développement des métastases est la cause principale de mortalité des patients atteints de cancer, mais demeure un obstacle majeur aux traitements. L'hypoxie, une caractéristique commune des tumeurs solides, est fortement impliquée dans l'invasion cellulaire et le développement des métastases, mais les mécanismes sous-jacents demeurent méconnus. La signalisation du LPA joue un rôle important au cours de la tumorigenèse et du développement des métastases, les membres de cette voie étant souvent régulés à la hausse dans les cellules tumorales. La signalisation du LPA a également été impliquée dans la production de structures de dégradation, les invadopodes, qui sont nécessaires à la formation de métastases. Des études récentes indiquent que la formation d'invadopodes est également induite par l'hypoxie. Par conséquent, nous avons voulu élucider l'influence du microenvironnement hypoxique sur l'axe de signalisation du LPA et si celui-ci joue un rôle dans la production d'invadopodes et la formation de métastases. Nous avons découvert que le LPA1 est un récepteur utilisé de façon commune et majoritaire pour la production d'invadopodes induite par l'hypoxie, et ce, dans diverses lignées cellulaires cancéreuses. Nous avons démontré que l'hypoxie favorise la formation d'invadopodes en utilisant une voie de signalisation distincte qui implique une communication croisée entre le récepteur LPA1 et l'EGFR, médiée par la kinase Src, Dans ce contexte, l'inhibition combinée du LPA1 et de l'EGFR agit en synergie afin d’empêcher la formation de métastases spontanées. Étant donné que la toxicité et la résistance aux inhibiteurs de l'EGFR représentent un défi important pour les patients atteints de cancer, ce travail permet l’identification d’une cible potentielle, le LPA1, qui pourrait être inhibée conjointement avec l'EGFR dans le but d’améliorer la survie de ces patients. D'autres études sur la régulation hypoxique de l'axe de signalisation du LPA ont démontré que l'hypoxie peut, Metastasis is the leading cause of cancer patient mortality yet remains a major hurdle for treatment. Hypoxia, a common feature of solid tumors, has been critically involved in cell invasion and metastasis but the underlying mechanisms remain poorly understood. The LPA signaling axis plays an important role during tumorigenesis and metastasis, with members of this pathway often being upregulated in tumor cells. LPA signaling has also been implicated in production of the degradative structures invadopodia, which are known to be required for metastasis. Interestingly, formation of invadopodia can also be induced by hypoxia. Therefore, we endeavoured to elucidate the influence of the hypoxic tumor microenvironment on the LPA signaling axis and whether this could play a role in invadopodia production and metastasis. We uncovered LPA1 as a common and major receptor used for hypoxia-induced invadopodia production in various cancer cell lines. We demonstrated that hypoxia promotes invadopodia formation through a distinct signaling pathway that involves Src-mediated cross-communication between LPA1 and EGFR, and that combined inhibition of LPA1 and EGFR acts synergistically to impede spontaneous metastasis. Since EGFR inhibitor toxicity and resistance represents a current challenge for cancer patients, this work identifies a potential target, LPA1 that could be inhibited in conjunction with EGFR to improve patient outcomes. Further studies into hypoxic regulation of the LPA signaling axis demonstrated that hypoxia can control the expression levels of LPA producing (ATX) and degrading (LPP1/LPP3) enzymes, events that lead to increased invadopodia production. Hypoxia was also found to alter the localization of these proteins, uncovering an additional mechanism of hypoxic regulation. Our work suggests that hypoxia is a master regulator of the LPA signaling axis that leads to metastasis, therefore therapies targeting this axis could be beneficial to counteract the de

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2015., Lysophosphatidic acid (LPA) is a lipid-signaling molecule with potent effects on cell growth, survival, and migration. In recent years, major progress has been made in deciphering the mechanisms of LPA generation and how it acts on target cells by binding to its specific receptors (named LPA1-6). The enzyme autotaxin generates LPA from the precursor lysophosphatidylcholine and is constitutively expressed in a number of tissues including high endothelial venules and the lung. In addition, autotaxin expression and activity are increased in different disease states, including asthma, which suggests that the autotaxin (ATX)/LPA axis may play a broader role in inflammation and immunity than previously thought. The work in this thesis is divided into two sections. First, we looked at the effects of LPA on T cell migration. We investigated the expression of the six LPA receptors on CD4+ T cells and studied the effects of LPA on T cell migration. We found that LPA induces T cell chemorepulsion and improves the quality of non-directed migration in vitro. In addition, we utilized intravital two-photon microscopy to visualize lymphocyte migration at HEVs and detected a striking defect in the early migratory behavior of lpa2 -/- T cells. We therefore concluded that LPA2 plays a previously unsuspected role in promoting T cell motility. The second part of this thesis project sought to determine the role of the ATX/LPA axis in promoting allergic airway inflammation in a murine model of allergeninduced asthma. We hypothesized that autotaxin is upregulated in lung epithelial cells during allergic inflammation and that it acts by generating LPA. To study this, we took three approaches. First, we used a novel LPA2 agonist, which activates a unique LPA receptor with anti-inflammatory properties, which protected mice from allergen-induced inflammation. Second, we tested an autotaxin inhibitor and found that it lessened the Th2 immune response after HDM challenge in mice. Thirdly, using

Background—Mendelian randomization studies have highlighted that lipoprotein(a) [Lp(a)] was associated with calcific aortic valve disease (CAVD). Lp(a) transports oxidized phospholipids (OxPLs) with a high content in lysophosphatidylcholine (LPC). Autotaxin (ATX) transforms LPC into lysophosphatidic acid. We hypothesized that ATX-lysophosphatidic acid could promote inflammation/mineralization of the aortic valve. Methods and Results—We have documented the expression of ATX in control and mineralized aortic valves. By using different approaches we have also investigated the role of ATX- lysophosphatidic acid on the mineralization of isolated valves interstitial cells (VICs) and in a mouse model of CAVD. Enzyme specific ATX activity was elevated by 60% in mineralized aortic valves compared to control valves. Immunohistochemistry studies showed a high level of ATX in mineralized aortic valves, which co-localized with OxPL and apolipoprotein(a). We detected a high level of ATX activity in the Lp(a) fraction in circulation. Interaction between ATX and Lp(a) was confirmed by in situ proximity ligation assay. Moreover, we documented that VICs also expressed ATX in CAVD. We showed that ATX-lysophosphatidic acid promote the mineralization of the aortic valve through a NF-¿B/IL-6/BMP2 pathway. In LDLR-/-/ApoB100/100/IGFII mice, ATX is overexpressed and lysophosphatidic acid promotes a strong deposition of hydroxyapatite of calcium in aortic valve leaflets and accelerates the development of CAVD. Conclusions—ATX is transported in the aortic valve by Lp(a) and is also secreted by VICs. ATX-lysophosphatidic acid promotes inflammation and mineralization of the aortic valve and thus could represent novel therapeutic targets in CAVD.

Background—Mendelian randomization studies have highlighted that lipoprotein(a) [Lp(a)] was associated with calcific aortic valve disease (CAVD). Lp(a) transports oxidized phospholipids (OxPLs) with a high content in lysophosphatidylcholine (LPC). Autotaxin (ATX) transforms LPC into lysophosphatidic acid. We hypothesized that ATX-lysophosphatidic acid could promote inflammation/mineralization of the aortic valve. Methods and Results—We have documented the expression of ATX in control and mineralized aortic valves. By using different approaches we have also investigated the role of ATX- lysophosphatidic acid on the mineralization of isolated valves interstitial cells (VICs) and in a mouse model of CAVD. Enzyme specific ATX activity was elevated by 60% in mineralized aortic valves compared to control valves. Immunohistochemistry studies showed a high level of ATX in mineralized aortic valves, which co-localized with OxPL and apolipoprotein(a). We detected a high level of ATX activity in the Lp(a) fraction in circulation. Interaction between ATX and Lp(a) was confirmed by in situ proximity ligation assay. Moreover, we documented that VICs also expressed ATX in CAVD. We showed that ATX-lysophosphatidic acid promote the mineralization of the aortic valve through a NF-¿B/IL-6/BMP2 pathway. In LDLR-/-/ApoB100/100/IGFII mice, ATX is overexpressed and lysophosphatidic acid promotes a strong deposition of hydroxyapatite of calcium in aortic valve leaflets and accelerates the development of CAVD. Conclusions—ATX is transported in the aortic valve by Lp(a) and is also secreted by VICs. ATX-lysophosphatidic acid promotes inflammation and mineralization of the aortic valve and thus could represent novel therapeutic targets in CAVD.

ObjectiveArticular cartilage is a highly specialized tissue which forms the surfaces in synovial joints. Full-thickness cartilage defects caused by trauma or microfracture surgery heal via the formation of fibrotic tissue characterized by a high content of collagen I (COL I) and subsequent poor mechanical properties. The goal of this study is to investigate the molecular mechanisms underlying fibrosis after joint injury.DesignRat knee joint models were used to mimic cartilage defects after acute injury. Immunohistochemistry was performed to detect proteins related to fibrosis. Human fetal chondrocytes and bone marrow stromal cells (BMSCs) were used to study the influence of the lipid lysophosphatidic acid (LPA) on COL I synthesis. Quantitative PCR, ELISA and immunohistochemistry were performed to evaluate the production of COL I. Chemical inhibitors were used to block LPA signaling both in vitro and in vivo.ResultsAfter full-thickness cartilage injury in rat knee joints, stromal cells migrating to the injury expressed high levels of the LPA-producing enzyme autotaxin (ATX); intact articular cartilage in rat and humans expressed negligible levels of ATX despite expressing the LPA receptors LPAR1 and LPAR2. LPA-induced increases in COL I production by chondrocytes and BMSCs were mediated by the MAP kinase and PI3 Kinase signaling pathways. Inhibition of the ATX/LPA axis significantly reduced COL I-enriched fibrocartilage synthesis in full-thickness cartilage defects in rats in favor of the collagen II-enriched normal state.ConclusionTaken together, these results identify an attractive target for intervention in reducing the progression of post-traumatic fibrosis and osteoarthritis.

BACKGROUND: Lysophosphatidic acid (LPA) is a bioactive phospholipid with a potentially causative role in neurotrauma. Blocking LPA signaling with the LPA-directed monoclonal antibody B3/Lpathomab is neuroprotective in the mouse spinal cord following injury. FINDINGS: Here we investigated the use of this agent in treatment of secondary brain damage consequent to traumatic brain injury (TBI). LPA was elevated in cerebrospinal fluid (CSF) of patients with TBI compared to controls. LPA levels were also elevated in a mouse controlled cortical impact (CCI) model of TBI and B3 significantly reduced lesion volume by both histological and MRI assessments. Diminished tissue damage coincided with lower brain IL-6 levels and improvement in functional outcomes. CONCLUSIONS: This study presents a novel therapeutic approach for the treatment of TBI by blocking extracellular LPA signaling to minimize secondary brain damage and neurological dysfunction.

La formation des métastases est une propriété fondamentale des cellules cancéreuses malignes ainsi que cause principale de décès chez les patients atteints de cancer. Des études récent indique que l'invasion tumorale et la formation des métastases peut être initié par la formation des protrusions riches en actine et capables de dégrader la matrice extracellulaire, appeles des invadopodes. Cependant, malgré les recherches importantes sur la biologie des invadopodes, les informations concernant les initiateurs spécifiques de ces structures lors de la progression tumorale demeurent limitées. L'autotaxin (ATX) est une lysophospholipase sécrétée dont les niveaux d'expression corrèlent avec l'agressivité et le potentiel invasif des tumeurs. L'ATX produit l'acide lysophosphatidique (LPA), un phospholipide impliqué dans la progression tumorale qui agit par l'intermédiaire de récepteurs couplés aux protéines G, LPAt-6. Il a été récemment démontré que la surexpression del' ATX et des récepteurs LPAt-3 cause une augmentation de l'invasion tumorale et de la formation de métastases in vivo, cependant, le rôle d'autres récepteurs, soit les LPA4-6, ainsi que les mécanismes exacts par lesquels l'ATX induit la formation de métastases demeurent peu connus. Afin d'étudier l'influence del' ATX sur la production d'invadopodes, nous avons transfecté des cellules de fibrosarcome, les HT-1080, avec des gène codant soit pour l 'ATX ou des ARN m interférant. Ces cellules ont été testées dans des essais de production d'invadopodes utilisant de la matrice fluorescente et des techniques d'immunofluorescence afm de visualiser de façon simultanée la dégradation de la matrice et les composantes caractéristiques de ces structures. Nos résultats indiquent que l 'ATX est impliquée dans la formation et les fonctions des invadopodes. Par l'ajout du LPC ou du LPA, le substrat et le produit de l' ATX, nous avons montré que la production d'invadopodes est dépendante de la production de LPA du LPC. Parmi l, Tumor metastasis is a fundamental property of malignant cancer cells and the major cause of death in cancer patients. Recent studies indicate that tumor cell invasion and metastasis may be initiated by the formation of the actin-rich cell protrusions with ECM degradation activity, invadopodia. However, despite extensive research on the biology of invadopodia, very little is known about their specific inducers during tumor progression. Autotaxin (ATX) is a secreted lysophospholipase whose expression levels within tumors correlates strongly with their aggressiveness and invasiveness. ATX produces lyosophosphatidic acid (LPA), a phospholipid with known tumor promoting functions that acts through the G-protein coupled receptors, LPA[subscript 1-6] . Recently, overexpression of ATX and LPA receptors (LPA[subscript 1-3]) has been linked to increased tumor invasion and metastasis in vivo , however, the role of other LPA receptors (LPA[subscript 4-6]) as well as the exact mechanisms by which ATX induces tumor metastasis remain poorly characterized. In order to determine the involvement of ATX and LPA in invadopodia production, we used the fibrosarcoma HT-1080 cells stably transfected with ATX or shRNA targeting ATX in fluorescent matrix degradation assays. Our results demonstrate that ATX is implicated in the production of invadopodia resulting in an increase in both their formation and function. Using LPC or LPA, the substrate and product of ATX, we further show that invadopodia production is dependent on the production of LPA from LPC. Among the LPA receptors, LPA 4 has the highest expression in HT1080 cells. Using LPA[subscript 4] shRNA as well as agonists and inhibitors of the cAMP pathway, we provide evidence that LPA[subscript 4] signaling through the cAMP-EPAC-Rap1 axis, regulates invadopodia formation downstream of ATX. Furthermore, inhibition of Rac1, a known effector of Rap1 and invadopodia formation, abolished EPAC-induced invadopodia production, sugges

The importance of tumor stroma in prostate cancer development and progression has been recognized through its stimulating effects on cancer cell growth, migration and angiogenesis. Our hypothesis was that the prostate cancer stroma is derived, at least in part, from mesenchymal stem cells (MSCs) that are proliferative, multipotent and self-renewing. Our objective was to demonstrate that cancer-derived stromal cells have characteristics of mesenchymal stem cells. Comparing primary cultures of stromal cells from cancer versus normal tissues, we observed (a) differential gene expression profiles, (b) increased expression of autotaxin, a motility factor, (c) increased expression of the MSC-associated antigen CD90, and (d) increased expression of transforming growth factor-beta, associated with tumor promotion, in CD90-positive cells. Many of these properties are consistent with a mesenchymal stem cell phenotype of prostate cancer-associated stroma, supporting our hypothesis.