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Yanfen Cui, Baoai Han, He Zhang, Hui Liu, Fei Zhang, Ruifang Niu Public Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Key Laboratory of Breast Cancer Prevention and Therapy, Ministry of Education, Tianjin, 300060, People’s Republic of ChinaCorrespondence: Ruifang Niu; Fei Zhang Tel +86 13752668992; +86 18622221365Email rniu@tmu.edu.cn; feizhang03@tmu.edu.cnBackground and Aim: Uncontrolled proliferation is the most prominent biological feature of tumors. In order to rapidly proliferate, tumor cells regulate their metabolic behavior by controlling the expression of metabolism-related genes (MRGs) to maximize the utilization of available nutrients. In this study, we aimed to construct prognosis models for colorectal adenocarcinoma (COAD) and rectum adenocarcinoma (READ) using MRGs to predict the prognoses of patients.Methods: We first acquired the gene expression profiles of COAD and READ from the TCGA database, and then utilized univariate Cox analysis, Lasso regression, and multivariable Cox analysis to identify the MRGs for risk models.Results: Eight genes (CPT1C, PLCB2, PLA2G2D, GAMT, ENPP2, PIP4K2B, GPX3, and GSR) in the colon cancer risk model and six genes (TDO2, PKLR, GAMT, EARS2, ACO1, and WAS) in the rectal cancer risk model were identified successfully. Multivariate Cox analysis indicated that these two models could accurately and independently predict overall survival (OS) for patients with COAD or READ. Furthermore, functional enrichment analysis was used to identify the metabolism pathway of MRGs in the risk models and analyzed these genes comprehensively. Then, we verified the prognosis model in independent COAD cohorts (GSE17538) and detected the correlations of the protein expression levels of GSR and ENPP2 with prognosis for COAD or READ.Conclusion: In this study, 14 MRGs were identified as potential prognostic biomarkers and therapeutic targets for colorectal cancer.Keywords: metabolism-related gene, colon adenocarcinoma, rectum adenocarcinoma, prognosis, ENPP2, GSR

Yanfen Cui, Baoai Han, He Zhang, Hui Liu, Fei Zhang, Ruifang Niu Public Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Key Laboratory of Breast Cancer Prevention and Therapy, Ministry of Education, Tianjin, 300060, People’s Republic of ChinaCorrespondence: Ruifang Niu; Fei Zhang Tel +86 13752668992; +86 18622221365Email rniu@tmu.edu.cn; feizhang03@tmu.edu.cnBackground and Aim: Uncontrolled proliferation is the most prominent biological feature of tumors. In order to rapidly proliferate, tumor cells regulate their metabolic behavior by controlling the expression of metabolism-related genes (MRGs) to maximize the utilization of available nutrients. In this study, we aimed to construct prognosis models for colorectal adenocarcinoma (COAD) and rectum adenocarcinoma (READ) using MRGs to predict the prognoses of patients.Methods: We first acquired the gene expression profiles of COAD and READ from the TCGA database, and then utilized univariate Cox analysis, Lasso regression, and multivariable Cox analysis to identify the MRGs for risk models.Results: Eight genes (CPT1C, PLCB2, PLA2G2D, GAMT, ENPP2, PIP4K2B, GPX3, and GSR) in the colon cancer risk model and six genes (TDO2, PKLR, GAMT, EARS2, ACO1, and WAS) in the rectal cancer risk model were identified successfully. Multivariate Cox analysis indicated that these two models could accurately and independently predict overall survival (OS) for patients with COAD or READ. Furthermore, functional enrichment analysis was used to identify the metabolism pathway of MRGs in the risk models and analyzed these genes comprehensively. Then, we verified the prognosis model in independent COAD cohorts (GSE17538) and detected the correlations of the protein expression levels of GSR and ENPP2 with prognosis for COAD or READ.Conclusion: In this study, 14 MRGs were identified as potential prognostic biomarkers and therapeutic targets for colorectal cancer.Keywords: metabolism-related gene, colon adenocarcinoma, rectum adenocarcinoma, prognosis, ENPP2, GSR

International audience; Inflammatory and neuropathic-like components underlie rheumatoid arthritis (RA)-associated pain, and lysophosphatidic acid (LPA) is linked to both joint inflammation in RA patients and to neuropathic pain. Thus, we investigated a role for LPA signalling using the collagen antibody-induced arthritis (CAIA) model. Pain-like behavior during the inflammatory phase and the late, neuropathic-like phase of CAIA was reversed by a neutralizing antibody generated against LPA and by an LPA1/3 receptor inhibitor, but joint inflammation was not affected. Autotaxin, an LPA synthesizing enzyme was upregulated in dorsal root ganglia (DRG) neurons during both CAIA phases, but not in joints or spinal cord. Late-phase pronociceptive neurochemical changes in the DRG were blocked in Lpar1 receptor deficient mice and reversed by LPA neutralization. In vitro and in vivo studies indicated that LPA regulates pain-like behavior via the LPA1 receptor on satellite glia cells (SGCs), which is expressed by both human and mouse SGCs in the DRG. Furthermore, CAIA-induced SGC activity is reversed by phospholipid neutralization and blocked in Lpar1 deficient mice. Our findings suggest that the regulation of CAIA-induced pain-like behavior by LPA signalling is a peripheral event, associated with the DRGs and involving increased pronociceptive activity of SGCs, which in turn act on sensory neurons.

Autotaxin (ATX) encoded by Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 (ENPP2) is a key enzyme in Lysophosphatidic Acid (LPA) synthesis implicated in cancer. Although its aberrant expression has been reported, ENPP2 methylation profiles in health and malignancy are not described. We examined in silico the methylation of ENPP2 analyzing publicly available methylome datasets, to identify Differentially Methylated CpGs (DMCs) which were then correlated with expression at gene and isoform levels. Significance indication was set to be FDR corrected p-value &lt, 0.05. Healthy tissues presented methylation in all gene body CGs and lower levels in Promoter Associated (PA) regions, whereas in the majority of the tumors examined (HCC, melanoma, CRC, LC and PC) the methylation pattern was reversed. DMCs identified in the promoter were located in sites recognized by multiple transcription factors, suggesting involvement in gene expression. Alterations in methylation were correlated to an aggressive phenotype in cancer cell lines. In prostate and lung adenocarcinomas, increased methylation of PA CGs was correlated to decreased ENPP2 mRNA expression and to poor prognosis parameters. Collectively, our results corroborate that methylation is an active level of ATX expression regulation in cancer. Our study provides an extended description of the methylation status of ENPP2 in health and cancer and points out specific DMCs of value as prognostic biomarkers.

Enpp2 is an enzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which exhibits a wide variety of biological functions. Here, we examined the biological effects of Enpp2 on dendritic cells (DCs), which are specialized antigen-presenting cells (APCs) characterized by their ability to migrate into secondary lymphoid organs and activate naïve T-cells. DCs were generated from bone marrow progenitors obtained from C57BL/6 mice. Enpp2 levels in DCs were regulated using small interfering (si)RNA or recombinant Enpp2. Expression of Enpp2 in LPS-stimulated mature (m)DCs was high, however, knocking down Enpp2 inhibited mDC function. In addition, the migratory capacity of mDCs increased after treatment with rmEnpp2, this phenomenon was mediated via the RhoA-mediated signaling pathway. Enpp2-treated mDCs showed a markedly increased capacity to migrate to lymph nodes in vivo. These findings strongly suggest that Enpp2 is necessary for mDC migration capacity, thereby increasing our understanding of DC biology. We postulate that regulating Enpp2 improves DC migration to lymph nodes, thus improving the effectiveness of cancer vaccines based on DC.

Anne-Li Lind,1 David Just,2 Maria Mikus,2 Claudia Fredolini,2 Marina Ioannou,2 Björn Gerdle,3 Bijar Ghafouri,3 Emmanuel Bäckryd,3 Lars Tanum,4 Torsten Gordh,1 Anna Månberg2 1Department of Surgical Sciences, Uppsala University, Uppsala, Sweden; 2Division of Affinity Proteomics, SciLifeLab, Deptartment of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden; 3Pain and Rehabilitation Center, and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; 4Department of R&D in Mental Health, Akershus University Hospital, Lørenskog, NorwayCorrespondence: Anna MånbergDivision of Affinity Proteomics, SciLifeLab, Department of Protein Science, KTH - Royal Institute of Technology, Tomtebodvägen 23A, Solna, Stockholm, SwedenTel +46 8 790 9803Email anna.manberg@scilifelab.seObjective: Neuropathic pain and fibromyalgia are two common and poorly understood chronic pain conditions that lack satisfactory treatments, cause substantial suffering and societal costs. Today, there are no biological markers on which to base chronic pain diagnoses, treatment choices or to understand the pathophysiology of pain for the individual patient. This study aimed to investigate cerebrospinal fluid(CSF) protein profiles potentially associated with fibromyalgia and neuropathic pain.Methods: CSF samples were collected from 25 patients with neuropathic pain (two independent sets, n=14 patients for discovery, and n=11 for verification), 40 patients with fibromyalgia and 134 controls without neurological disease from two different populations. CSF protein profiling of 55 proteins was performed using antibody suspension bead array technology.Results: We found increased levels of apolipoprotein C1 (APOC1) in CSF of neuropathic pain patients compared to controls and there was a trend for increased levels also in fibromyalgia patients. In addition, levels of ectonucleotide pyrophosphatase family member 2 (ENPP2, also referred to as autotaxin) were increased in the CSF of fibromyalgia patients compared to all other groups including patients with neuropathic pain.Conclusion: The increased levels of APOC1 and ENPP2 found in neuropathic pain and fibromyalgia patients may shed light on the underlying mechanisms of these conditions. Further investigation is required to elucidate their role in maintaining pain and other main symptoms of these disorders.Keywords: cerebrospinal fluid, neuropathic pain, fibromyalgia, antibody suspension bead arrays, APOC1, ENPP2 &nbsp

Autotaxin (ATX) converts lysophosphatidylcholine and sphingosyl-phosphorylcholine into lysophosphatidic acid and sphingosine 1-phosphate, respectively. Despite the pivotal function of ATX in lipid metabolism, mechanisms by which ATX regulates immune and inflammatory disorders remain elusive. Here, using myeloid cell lineage-restricted Atx knockout mice, we show that Atx deficiency disrupts membrane microdomains and lipid rafts, resulting in the inhibition of Toll-like receptor 4 (TLR4) complex formation and the suppression of adaptor recruitment, thereby inhibiting TLR4-mediated responses in macrophages. Accordingly, TLR4-induced innate immune functions, including phagocytosis and iNOS expression, are attenuated in Atx-deficient macrophages. Consequently, Atx-/- mice exhibit a higher bacterial prevalence in the intestinal mucosa compared to controls. When combined with global Il10-/- mice, which show spontaneous colitis due to the translocation of luminal commensal microbes into the mucosa, myeloid cell lineage-restricted Atx knockout accelerates colitis development compared to control littermates. Collectively, our data reveal that Atx deficiency compromises innate immune responses, thereby promoting microbe-associated gut inflammation.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare disease, with an epidemiology similar to that of pulmonary arterial hypertension (PAH). CTEPH is a subgroup of pulmonary hypertension (PH) with a cumulative incidence between 0.1% and 9.1% in patients with reported pulmonary embolism. The state-of-the-art therapeutic strategy is pulmonary endarterectomy (PEA). Yet, approximately 60% of the patients are eligible for surgery and significant number of patients develop persistent/recurrent PH after PEA. Furthermore, around 25% of patients with subsegmental obstruction are candidates for balloon angioplasty (BPA), and/or medical therapy, developed and used in the past few years. Hence, better understanding of the pathophysiology and molecular mechanisms driving the CTEPH is mandatory in order to identify effective treatments especially for patients with absence of proximal obliteration. The existence of microvascular disease in CTEPH, indistinguishable from idiopathic PAH (IPAH) was at first acknowledged by Moser and Bloor in a lung tissues obtained from biopsy or at autopsy of CTEPH patients. On one hand, the vascular lesions exhibited eccentric intimal fibrosis, intimal fibromuscular proliferation, medial hypertrophy and presence of plexiform lesions. On the other hand, it was postulated that the vascular remodelling distal to the thromboembolic obstruction may be similarly distributed in the lung areas of complete and non-complete occlusion CTEPH. Based on prior research, the following research questions are of interest to better understand the CTEPH: (1) Are the molecular and histological imprints of CTEPH comparable or distinct to the extensively studied PAH? (2) Are there significant differences from histological or molecular aspect between the vascular lesions in proximal (central) and distal (peripheral) CTEPH? (3) Is the small vessel arteriopathy homogeneously distributed in different lobes, irrespective of the site of the disease? (4) Are the lung biopsies from patients who develop PH due to sarcoma resembling the “classical” histopathological changes observed in CTEPH? (5) What is the functional role of the unbiasedly selected targets in terms of development or maintenance of CTEPH? In order to answer these research questions, this thesis aim to histologically and molecularly characterize the end-stage CTEPH enfolding several facets and further, the ongoing CTEPH in terms of central and occluded (completely and non-completely) vascular lesions. First, multi-centre human samples have been collected and investigated to identify whether vascular remodelling in the end-stage CTEPH is as similar as in the other groups of PH, esp. in the well-studied idiopathic PAH group. Different morphometric approaches were applied to assess the vascular remodelling. Next, the analysis of global transcriptional landscape in CTEPH and IPAH was performed from laser capture microdissected vessels, to identify the common or the unique mechanisms driving small vessel arteriopathy in CTEPH as compared to IPAH. These findings suggest that the distal vascular remodelling in CTEPH, resemble vascular remodelling in IPAH. Furthermore, CTEPH manifests significantly divergent global transcriptional landscape in comparison to IPAH. Second, the results show that central manifest similar histological changes as peripheral CTEPH, while the global transcriptional landscape of central CTEPH manifests similar and unique signaling pathways in contrast to the peripheral CTEPH. Third, the results show that small vessel arteriopathy is homogeneously distributed in different lobes, irrespective of the site of the disease. Forth, similarly patients who develop PH due to sarcoma and underwent PEA present comprehensive vascular remodelling, similar to end-stage CTEPH patients. Fifth, it is revealed that CHI3L1 and ENPP2, differently regulated genes between CTEPH and IPAH, and similarly regulated in proximal and distal CTEPH, are involved in pulmonary vascular remodelling in vitro and ex vivo via regulation of pro-proliferative, pro-migratory and pro-contractile mechanisms, suggesting a potential contribution to the pathogenesis of CTEPH. Taken together, further studies are needed to understand how exact factors, such as high share stress, hypoxia or inflammatory molecules trigger the interlaying pathological and molecular mechanisms in CTEPH. Chronische thromboembolische pulmonale Hypertonie (CTEPH) ist eine seltene Krankheit mit einer Epidemiologie, die der pulmonalen arteriellen Hypertonie (PAH) ähnelt. CTEPH ist eine Untergruppe der pulmonalen Hypertonie (PH), die zwischen 0,1% und 9,1% bei Patienten mit einer gemeldeter Lungenembolie auftritt. Die beste bekannte Therapiestrategie ist die pulmonale Endarteriektomie (PEA). Allerdings kommen nur etwa 60% der Patienten für eine Operation in Frage und eine signifikant höhere Anzahl von Patienten entwickelt eine persistierende/rezidivierende PH nach der PEA. Weiterhin sind rund 25% der Patienten mit subsegmentaler Obstruktion Kandidaten für eine Ballonangioplastie (BPA) und/oder eine medizinische Therapie, die in den letzten Jahren entwickelt und angewendet wurde. Daher ist ein besseres Verständnis der Pathophysiologie und der molekularen Mechanismen, die die Entwicklung von CTEPH treiben und beeinflussen, unabdingbar, um wirksame alternative Therapien zu identifizieren. Dies gilt insbesondere für Patienten ohne proximale Obliteration. Kleingefäßerkrankungen bei CTEPH identisch zur idiopathischen PAH (IPAH) wurden als erstes von Moser und Bloor in durch Biopsie oder Autopsie von Patienten gewonnenem Lungengewebe nachgewiesen. Einerseits zeigten die Gefäßläsionen eine exzentrische Intimalfibrose, mediale Hypertrophie sowie das Vorhandensein plexiformer Läsionen. Andererseits wurde postuliert, dass die vaskuläre Remodellierung distal zu der thromboembolischen Obstruktion eine ähnliche Verteilung in den Lungenbereichen der vollständigen und nicht vollständigen CTEPH-Okklusion aufweist. Basierend auf vorheriger Forschung sind die folgenden Fragen für ein besseres Verständnis von CTEPH von Interesse: (1) Sind die molekularen und histologischen Abdrücke von CTEPH mit denen der intensiv untersuchten PAH vergleichbar oder verschieden? (2) Gibt es signifikante Unterschiede aus histologischer oder molekularer Perspektive zwischen den Gefäßläsionen bei proximaler (zentraler) und distaler (peripherer) CTEPH? (3) Ist die kleine Gefäßarteriopathie unabhängig vom Ort der Erkrankung homogen in verschiedenen Lappen verteilt? (4) Sind die Lungenbiopsien von Patienten, die aufgrund eines Sarkoms eine PH entwickeln, den bei CTEPH beobachteten „klassischen“ histopathologischen Veränderungen ähnlich? (5) Welche funktionale Rolle spielen die zufällig ausgewählten Ziele bei der Entwicklung oder dem Bestehen von CTEPH? Um diese Fragen zu beantworten, hat die vorliegende Arbeit das Ziel CTPEH im Endstadium histologisch und molekular aus verschiedenen Sichtweisen zu charakterisieren. Darüber hinaus soll eine bestehende CTEPH in Bezug auf zentrale und verschlossene (vollständig und nicht vollständig) Gefäßläsionen untersucht werden. Erstens wurden multizentrische humane Proben gesammelt und untersucht, ob die vaskuläre Remodellierung im CTEPH-Endstadium ähnlich zu anderen Gruppen von PH ist, insbesondere in der extensiv untersuchten Gruppe der idiopathischen PAH. Dazu wurden verschiedene morphometrische Ansätze wie die mediale Wandstärke, das Neointima / Media-Verhältnis und die Kollagenablagerung genutzt, um die Remodellierung der Gefäße zu messen und zu bewerten. Weiterhin wurden die globalen Transkriptionslandschaften in CTEPH und IPAH an mikrodissezierten Gefäßen mit Lasereinfang analysiert. Anschließend wurden Microarrays und eine bioinformatische Analyse durchgeführt, um die gemeinsamen oder einzigartigen Mechanismen zu identifizieren, die die Arteriopathie kleiner Gefäße in CTEPH im Vergleich zu IPAH treiben. Diese Ergebnisse legen nahe, dass die distale Gefäßumbildung bei CTEPH derjenigen bei IPAH ähnelt. Darüber hinaus weist CTEPH im Vergleich zu IPAH eine signifikant abweichende globale Transkriptionslandschaft auf. Zweitens zeigen die Ergebnisse, dass die zentrale ähnliche histologische Veränderungen aufweist wie die periphere CTEPH. Allerdings zeigt die globale Transkriptionsregulationslandschaft der zentralen CTEPH im Gegensatz zur peripheren CTEPH zwar ähnliche aber auch einzigartige Regulation der Signalwege. Drittens hat sich gezeigt, dass die Arteriopathie der kleinen Gefäße unabhängig vom Ort der Erkrankung homogen in verschiedenen Lappen verteilt ist. Interessanterweise zeigten sowohl die vorher als auch hinterher betroffene Stelle eine ähnliche Verteilung der Intensität der Gefäßumbildung. Viertens, weisen Patienten, die aufgrund eines Sarkoms eine PH entwickeln sowie eine PEA durchlaufen haben, eine umfassende Gefäßumbildung ähnlich wie CTEPH-Patienten im Endstadium auf. Fünftens zeigt sich, dass CHI3L1 und ENPP2, unterschiedlich regulierte Gene zwischen CTEPH und IPAH und ähnlich reguliert in proximaler und distaler CTEPH, in vitro und ex vivo an der Remodellierung der Lungengefäße beteiligt sind. Dies erfolgt durch positive Wachstums-, Migrations- und Kontraktile-Mechanismen, die auf einen möglichen Beitrag zur Pathogenese von CTEPH hindeuten.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare disease, with an epidemiology similar to that of pulmonary arterial hypertension (PAH). CTEPH is a subgroup of pulmonary hypertension (PH) with a cumulative incidence between 0.1% and 9.1% in patients with reported pulmonary embolism. The state-of-the-art therapeutic strategy is pulmonary endarterectomy (PEA). Yet, approximately 60% of the patients are eligible for surgery and significant number of patients develop persistent/recurrent PH after PEA. Furthermore, around 25% of patients with subsegmental obstruction are candidates for balloon angioplasty (BPA), and/or medical therapy, developed and used in the past few years. Hence, better understanding of the pathophysiology and molecular mechanisms driving the CTEPH is mandatory in order to identify effective treatments especially for patients with absence of proximal obliteration. The existence of microvascular disease in CTEPH, indistinguishable from idiopathic PAH (IPAH) was at first acknowledged by Moser and Bloor in a lung tissues obtained from biopsy or at autopsy of CTEPH patients. On one hand, the vascular lesions exhibited eccentric intimal fibrosis, intimal fibromuscular proliferation, medial hypertrophy and presence of plexiform lesions. On the other hand, it was postulated that the vascular remodelling distal to the thromboembolic obstruction may be similarly distributed in the lung areas of complete and non-complete occlusion CTEPH. Based on prior research, the following research questions are of interest to better understand the CTEPH: (1) Are the molecular and histological imprints of CTEPH comparable or distinct to the extensively studied PAH? (2) Are there significant differences from histological or molecular aspect between the vascular lesions in proximal (central) and distal (peripheral) CTEPH? (3) Is the small vessel arteriopathy homogeneously distributed in different lobes, irrespective of the site of the disease? (4) Are the lung biopsies from patients who develop PH due to sarcoma resembling the classical histopathological changes observed in CTEPH? (5) What is the functional role of the unbiasedly selected targets in terms of development or maintenance of CTEPH?In order to answer these research questions, this thesis aim to histologically and molecularly characterize the end-stage CTEPH enfolding several facets and further, the ongoing CTEPH in terms of central and occluded (completely and non-completely) vascular lesions. First, multi-centre human samples have been collected and investigated to identify whether vascular remodelling in the end-stage CTEPH is as similar as in the other groups of PH, esp. in the well-studied idiopathic PAH group. Different morphometric approaches were applied to assess the vascular remodelling. Next, the analysis of global transcriptional landscape in CTEPH and IPAH was performed from laser capture microdissected vessels, to identify the common or the unique mechanisms driving small vessel arteriopathy in CTEPH as compared to IPAH. These findings suggest that the distal vascular remodelling in CTEPH, resemble vascular remodelling in IPAH. Furthermore, CTEPH manifests significantly divergent global transcriptional landscape in comparison to IPAH.Second, the results show that central manifest similar histological changes as peripheral CTEPH, while the global transcriptional landscape of central CTEPH manifests similar and unique signaling pathways in contrast to the peripheral CTEPH. Third, the results show that small vessel arteriopathy is homogeneously distributed in different lobes, irrespective of the site of the disease. Forth, similarly patients who develop PH due to sarcoma and underwent PEA present comprehensive vascular remodelling, similar to end-stage CTEPH patients. Fifth, it is revealed that CHI3L1 and ENPP2, differently regulated genes between CTEPH and IPAH, and similarly regulated in proximal and distal CTEPH, are involved in pulmonary vascular remodelling in vitro and ex vivo via regulation of pro-proliferative, pro-migratory and pro-contractile mechanisms, suggesting a potential contribution to the pathogenesis of CTEPH.Taken together, further studies are needed to understand how exact factors, such as high share stress, hypoxia or inflammatory molecules trigger the interlaying pathological and molecular mechanisms in CTEPH., Chronische thromboembolische pulmonale Hypertonie (CTEPH) ist eine seltene Krankheit mit einer Epidemiologie, die der pulmonalen arteriellen Hypertonie (PAH) ähnelt. CTEPH ist eine Untergruppe der pulmonalen Hypertonie (PH), die zwischen 0,1% und 9,1% bei Patienten mit einer gemeldeter Lungenembolie auftritt. Die beste bekannte Therapiestrategie ist die pulmonale Endarteriektomie (PEA). Allerdings kommen nur etwa 60% der Patienten für eine Operation in Frage und eine signifikant höhere Anzahl von Patienten entwickelt eine persistierende/rezidivierende PH nach der PEA. Weiterhin sind rund 25% der Patienten mit subsegmentaler Obstruktion Kandidaten für eine Ballonangioplastie (BPA) und/oder eine medizinische Therapie, die in den letzten Jahren entwickelt und angewendet wurde. Daher ist ein besseres Verständnis der Pathophysiologie und der molekularen Mechanismen, die die Entwicklung von CTEPH treiben und beeinflussen, unabdingbar, um wirksame alternative Therapien zu identifizieren. Dies gilt insbesondere für Patienten ohne proximale Obliteration. Kleingefäßerkrankungen bei CTEPH identisch zur idiopathischen PAH (IPAH) wurden als erstes von Moser und Bloor in durch Biopsie oder Autopsie von Patienten gewonnenem Lungengewebe nachgewiesen. Einerseits zeigten die Gefäßläsionen eine exzentrische Intimalfibrose, mediale Hypertrophie sowie das Vorhandensein plexiformer Läsionen. Andererseits wurde postuliert, dass die vaskuläre Remodellierung distal zu der thromboembolischen Obstruktion eine ähnliche Verteilung in den Lungenbereichen der vollständigen und nicht vollständigen CTEPH-Okklusion aufweist.Basierend auf vorheriger Forschung sind die folgenden Fragen für ein besseres Verständnis von CTEPH von Interesse: (1) Sind die molekularen und histologischen Abdrücke von CTEPH mit denen der intensiv untersuchten PAH vergleichbar oder verschieden? (2) Gibt es signifikante Unterschiede aus histologischer oder molekularer Perspektive zwischen den Gefäßläsionen bei proximaler (zentraler) und distaler (peripherer) CTEPH? (3) Ist die kleine Gefäßarteriopathie unabhängig vom Ort der Erkrankung homogen in verschiedenen Lappen verteilt? (4) Sind die Lungenbiopsien von Patienten, die aufgrund eines Sarkoms eine PH entwickeln, den bei CTEPH beobachteten klassischen histopathologischen Veränderungen ähnlich? (5) Welche funktionale Rolle spielen die zufällig ausgewählten Ziele bei der Entwicklung oder dem Bestehen von CTEPH?Um diese Fragen zu beantworten, hat die vorliegende Arbeit das Ziel CTPEH im Endstadium histologisch und molekular aus verschiedenen Sichtweisen zu charakterisieren. Darüber hinaus soll eine bestehende CTEPH in Bezug auf zentrale und verschlossene (vollständig und nicht vollständig) Gefäßläsionen untersucht werden.Erstens wurden multizentrische humane Proben gesammelt und untersucht, ob die vaskuläre Remodellierung im CTEPH-Endstadium ähnlich zu anderen Gruppen von PH ist, insbesondere in der extensiv untersuchten Gruppe der idiopathischen PAH. Dazu wurden verschiedene morphometrische Ansätze wie die mediale Wandstärke, das Neointima / Media-Verhältnis und die Kollagenablagerung genutzt, um die Remodellierung der Gefäße zu messen und zu bewerten. Weiterhin wurden die globalen Transkriptionslandschaften in CTEPH und IPAH an mikrodissezierten Gefäßen mit Lasereinfang analysiert. Anschließend wurden Microarrays und eine bioinformatische Analyse durchgeführt, um die gemeinsamen oder einzigartigen Mechanismen zu identifizieren, die die Arteriopathie kleiner Gefäße in CTEPH im Vergleich zu IPAH treiben. Diese Ergebnisse legen nahe, dass die distale Gefäßumbildung bei CTEPH derjenigen bei IPAH ähnelt. Darüber hinaus weist CTEPH im Vergleich zu IPAH eine signifikant abweichende globale Transkriptionslandschaft auf.Zweitens zeigen die Ergebnisse, dass die zentrale ähnliche histologische Veränderungen aufweist wie die periphere CTEPH. Allerdings zeigt die globale Transkriptionsregulationslandschaft der zentralen CTEPH im Gegensatz zur peripheren CTEPH zwar ähnliche aber auch einzigartige Regulation der Signalwege.Drittens hat sich gezeigt, dass die Arteriopathie der kleinen Gefäße unabhängig vom Ort der Erkrankung homogen in verschiedenen Lappen verteilt ist. Interessanterweise zeigten sowohl die vorher als auch hinterher betroffene Stelle eine ähnliche Verteilung der Intensität der Gefäßumbildung. Viertens, weisen Patienten, die aufgrund eines Sarkoms eine PH entwickeln sowie eine PEA durchlaufen haben, eine umfassende Gefäßumbildung ähnlich wie CTEPH-Patienten im Endstadium auf.Fünftens zeigt sich, dass CHI3L1 und ENPP2, unterschiedlich regulierte Gene zwischen CTEPH und IPAH und ähnlich reguliert in proximaler und distaler CTEPH, in vitro und ex vivo an der Remodellierung der Lungengefäße beteiligt sind. Dies erfolgt durch positive Wachstums-, Migrations- und Kontraktile-Mechanismen, die auf einen möglichen Beitrag zur Pathogenese von CTEPH hindeuten.

Autotaxin (ATX), encoded by the ctonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) gene, is a key enzyme in lysophosphatidic acid (LPA) synthesis. We have recently described ENPP2 methylation profiles in health and multiple malignancies and demonstrated correlation to its aberrant expression. Here we focus on breast cancer (BrCa), analyzing in silico publicly available BrCa methylome datasets, to identify differentially methylated CpGs (DMCs) and correlate them with expression. Numerous DMCs were identified between BrCa and healthy breast tissues in the gene body and promoter-associated regions (PA). PA DMCs were upregulated in BrCa tissues in relation to normal, in metastatic BrCa in relation to primary, and in stage I BrCa in relation to normal, and this was correlated to decreased mRNA expression. The first exon DMC was also investigated in circulating cell free DNA (ccfDNA) isolated by BrCa patients; methylation was increased in BrCa in relation to ccfDNA from healthy individuals, confirming in silico results. It also differed between patient groups and was correlated to the presence of multiple metastatic sites. Our data indicate that promoter methylation of ENPP2 arrests its transcription in BrCa and introduce first exon methylation as a putative biomarker for diagnosis and monitoring which can be assessed in liquid biopsy.

Anne-Li Lind,1 David Just,2 Maria Mikus,2 Claudia Fredolini,2 Marina Ioannou,2 Björn Gerdle,3 Bijar Ghafouri,3 Emmanuel Bäckryd,3 Lars Tanum,4 Torsten Gordh,1 Anna Månberg2 1Department of Surgical Sciences, Uppsala University, Uppsala, Sweden; 2Division of Affinity Proteomics, SciLifeLab, Deptartment of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden; 3Pain and Rehabilitation Center, and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; 4Department of R&D in Mental Health, Akershus University Hospital, Lørenskog, NorwayCorrespondence: Anna MånbergDivision of Affinity Proteomics, SciLifeLab, Department of Protein Science, KTH - Royal Institute of Technology, Tomtebodvägen 23A, Solna, Stockholm, SwedenTel +46 8 790 9803Email anna.manberg@scilifelab.seObjective: Neuropathic pain and fibromyalgia are two common and poorly understood chronic pain conditions that lack satisfactory treatments, cause substantial suffering and societal costs. Today, there are no biological markers on which to base chronic pain diagnoses, treatment choices or to understand the pathophysiology of pain for the individual patient. This study aimed to investigate cerebrospinal fluid(CSF) protein profiles potentially associated with fibromyalgia and neuropathic pain.Methods: CSF samples were collected from 25 patients with neuropathic pain (two independent sets, n=14 patients for discovery, and n=11 for verification), 40 patients with fibromyalgia and 134 controls without neurological disease from two different populations. CSF protein profiling of 55 proteins was performed using antibody suspension bead array technology.Results: We found increased levels of apolipoprotein C1 (APOC1) in CSF of neuropathic pain patients compared to controls and there was a trend for increased levels also in fibromyalgia patients. In addition, levels of ectonucleotide pyrophosphatase family member 2 (ENPP2, also referred to as autotaxin) were increased in the CSF of fibromyalgia patients compared to all other groups including patients with neuropathic pain.Conclusion: The increased levels of APOC1 and ENPP2 found in neuropathic pain and fibromyalgia patients may shed light on the underlying mechanisms of these conditions. Further investigation is required to elucidate their role in maintaining pain and other main symptoms of these disorders.Keywords: cerebrospinal fluid, neuropathic pain, fibromyalgia, antibody suspension bead arrays, APOC1, ENPP2 &nbsp

BACKGROUND/AIMS:Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.METHODS:Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.RESULTS:Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.CONCLUSION:Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors. Fil: Pleli, Thomas. Goethe Universitat Frankfurt; Alemania Fil: Mondorf, Antonia. Goethe Universitat Frankfurt; Alemania Fil: Ferreiros, Nerea. Goethe Universitat Frankfurt; Alemania Fil: Thomas, Dominique. Goethe Universitat Frankfurt; Alemania Fil: Dvorak, Karel. Goethe Universitat Frankfurt; Alemania Fil: Biondi, Ricardo Miguel. Goethe Universitat Frankfurt; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Heringdorf, Dagmar Meyer zu. Goethe Universitat Frankfurt; Alemania Fil: Zeuzem, Stefan. Goethe Universitat Frankfurt; Alemania Fil: Geisslinger, Gerd. Goethe Universitat Frankfurt; Alemania Fil: Zimmermann, Herbert. Goethe Universitat Frankfurt; Alemania Fil: Waidmann, Oliver. Goethe Universitat Frankfurt; Alemania Fil: Piiper, Albrecht. Goethe Universitat Frankfurt; Alemania

Inhibition of Autotaxin (ATX) is a potential treatment strategy for several diseases, including tumors with elevated ATX-lysophosphatidic acid (LPA) signaling. Combining structure-based virtual screening together with hen egg-white Autotaxin (ewATX) activity assays enabled the discovery of novel small-molecule ATX inhibitors with a 2,4-dihydropyrano[2,3-c]pyrazole scaffold. These compounds are suggested to bind to the lipophilic pocket, leaving the active site unrestrained. Our most potent compound, (S)-6-amino-4-(3,4-dichlorophenyl)-3-(4-[(4-fluorobenzyl)oxy]phenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile [(S)-25], inhibited human ATX (hATX) with an IC50-value of 134 nM. It also blocked ATX-evoked but not LPA-mediated A2058 melanoma cell migration. Noteworthy, molecular modeling correctly predicted the biologically active enantiomer of 2,4-dihydropyrano[2,3-c]pyrazoles, as verified by compound crystallization and activity assays. Our study established the ewATX activity assay as a valid and affordable tool in ATX inhibitor discovery. Overall, our study offers novel insights and approaches into design of novel ATX inhibitors targeting the hydrophobic pocket instead of the active site., final draft, peerReviewed

Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses.

Systematic ablation of previously identified dermal papilla (DP) signature genes in embryonic DP precursors will reveal their functional roles during hair follicle morphogenesis. In this study, we validate Enpp2/Autotaxin as one of the highest expressed signature genes in postnatal DP, and demonstrate specific expression of this lysophosphatidic acid (LPA)–generating enzyme in embryonic dermal condensates. We further identify dermal and epidermal expression of several LPA receptors, suggesting that LPA signaling could contribute to follicle morphogenesis in both mesenchymal and epithelial compartments. We then use the recently characterized Cre-expressing Tbx18 knock-in line to conditionally ablate Enpp2 in embryonic DP precursors. Despite efficient gene knockout in embryonic day 14.5 (E14.5) dermal condensates, morphogenesis proceeds regularly with normal numbers, lengths, and sizes of all hair follicle types, suggesting that Enpp2 is not required for hair follicle formation. To interrogate DP signature gene expression, we finally isolate control and Enpp2-null DP precursors and identify the expression and upregulation of LIPH, an alternative LPA-producing enzyme, suggesting that this gene could functionally compensate for the absence of Enpp2. We conclude that future coablation of both LPA-producing enzymes or of several LPA receptors may reveal the functional role of LPA signaling during hair follicle morphogenesis.

Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses.

Ectonucleotide pyrophosphatase/phosphodiestrase 2 (Enpp2) isolated from the supernatant of human melanoma cells is a lysophospholipase D that transforms lysophosphatidylcholine into lysophospatidic acid. Although multiple analyses have investigated the function of Enpp2 in the hypothalamus, its role in the uterus during the estrous cycle is not well understood. In the present study, rat uterine Enpp2 was analyzed by RT-PCR, Western blotting, and immunohistochemistry. Quantitative PCR analysis demonstrated that uterine Enpp2 mRNA was decreased during estrus compared to proestrus and diestrus. To determine whether uterine Enpp2 expression is affected by sex steroid hormones, immature rats were treated with 17β-estradiol (E2), progesterone, or both on postnatal days 14 to 16. Interestingly, the expression of Enpp2 mRNA and protein were down-regulated by E2 in the uterus during estrus but not during proestrus or diestrus, suggesting that Enpp2 may play a role in uterine function during estrus. Enpp2 is primarily localized in the stromal cells of the endometrium during proestrus and estrus. During diestrus, Enpp2 was highly expressed in the epithelial cells of the endometrium. Taken together, these results suggest that uterine Enpp2 may be regulated by E2 and plays a role in reproductive functions during female rat development.

Autotaxin, a four-domain ∼100 kDa mammalian glycoprotein, was expressed in stably transfected mammalian cells, purified from the medium and crystallized. Diffraction data from micrometre-thick crystal plates were collected on various European synchrotron beamlines and are presented and analysed., Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an ∼100 kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0 Å resolution and allowed the collection of complete diffraction data to about 2.6 Å resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed.

Background-Vascular smooth muscle cells (SMC), which are a major component of the atherosclerotic lesions, have an important role in initiation and progression of the atherosclerotic process. VSMCs migrate from the media into the intima layer of the arterial wall where they switch from a contractile to a synthetic phenotype, causing intimal thickening and plaque progression. Aim- The major aims of this study were 1)to reproducibly isolate and thoroughly characterize human arterial SMC subpopulations in order to evaluate their role in atheroma formation and their phenotypic changes associated to the atherosclerotic process; 2)to analyze the expression profiles of SMC subpopulations, in particular to validate signals stemming from a previous microarray study and 3) to investigate these signals both at RNA and protein level, on cultured SMCs cells and on carotid tissues directly. Methods-The following methods were used: 1)primary cell cultures of SMCs from normal (NP) and diseased (DP) portions of human carotid artery by tissue explantation; 2) quantitative real-time PCR (qPCR) on cultured VSMCs; 3)immunofluorescence (IF) on cultured VSMCs and carotid specimens 4) immunohistochemistry (IHC) on carotid specimens. Results- This study shows that large and small SMC subpopulations with different phenotypes can be isolated form CEA. Characterization of both subpopulations indicates that small SMCs exhibit "atheroma-prone" phenotype, characterized by high proliferation and low level of differentiation. We show that small SMCs, overexpressing CaM, proliferate more actively than large SMCs. Coculture of large SMCs with plaque-derived macrophages or conditioned medium or plaque induced a transition to the small phenotype with increased CaM expression. The CaM inhibitor W-7 decreased the proliferation of small SMCs and partially prevented the large to small phenotypic transition. Analysis of the expression profiles in SMC subpopulations and in carotid tissue specimens showed differences, which point to NOV (which belongs to CCN family) and ENPP2 (the enzyme that catalyzes the production of LPA) as potential components of the athero-prone phenotype. Additional studies in a larger number of samples, both at RNA and protein levels in cells and in tissue are needed to confirm data obtained. Moreover experiments addressing functional implications would required to corroborate the suggested association of markers, like CaM, NOV and ENPP2, with the atherosclerotic process.

Background Epidemiologic studies and animal models suggest that in utero arsenic exposure affects fetal health, with a negative association between maternal arsenic ingestion and infant birth weight often observed. However, the molecular mechanisms for this association remain elusive. In the present study, we aimed to increase our understanding of the impact of low-dose arsenic exposure on fetal health by identifying possible arsenic-associated fetal tissue biomarkers in a cohort of pregnant women exposed to arsenic at low levels. Methods Arsenic concentrations were determined from the urine samples of a cohort of 133 pregnant women from New Hampshire. Placental tissue samples collected from enrollees were homogenized and profiled for gene expression across a panel of candidate genes, including known arsenic regulated targets and genes involved in arsenic transport, metabolism, or disease susceptibility. Multivariable adjusted linear regression models were used to examine the relationship of candidate gene expression with arsenic exposure or with birth weight of the baby. Results Placental expression of the arsenic transporter AQP9 was positively associated with maternal urinary arsenic levels during pregnancy (coefficient estimate: 0.25; 95% confidence interval: 0.05 – 0.45). Placental expression of AQP9 related to expression of the phospholipase ENPP2 which was positively associated with infant birth weight (coefficient estimate: 0.28; 95% CI: 0.09 – 0.47). A structural equation model indicated that these genes may mediate arsenic’s effect on infant birth weight (coefficient estimate: -0.009; 95% confidence interval: -0.032 – -0.001; 10,000 replications for bootstrapping). Conclusions We identified the expression of AQP9 as a potential fetal biomarker for arsenic exposure. Further, we identified a positive association between the placental expression of phospholipase ENPP2 and infant birth weight. These findings suggest a path by which arsenic may affect birth outcomes.