Mixed hepatocellular cholangiocarcinoma tumors: Cholangiolocellular carcinoma is a distinct molecular entity

Background & Aims: Mixed hepatocellular cholangiocarcinoma (HCC-CCA) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy.Methods: Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 18 patients with mixed HCC-CCA were performed, encompassing the whole histological spectrum of the disease. Comparative genomic analysis was carried out, using independent datasets of HCC (n = 164) and intrahepatic cholangiocarcinoma (iCCA) (n = 149).Results: Integrative genomic analysis of HCC-CCAs revealed that cholangiolocellular carcinoma (CLC) represents a distinct biliaryderived entity compared with the stem-cell and classical types. CLC tumors were neural cell adhesion molecule (NCAM) positive (6/6 vs. 1/12, p < 0.001), chromosomally stable (mean chromosomal aberrations 5.7 vs. 14.1, p = 0.008), showed significant upregulation of transforming growth factor (TGF)-beta signaling and enrichment of inflammation-related and immune response signatures (p < 0.001). Stem-cell tumors were characterized by spaltlike transcription factor 4 (SALL4) positivity (6/8 vs. 0/10, p < 0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., MYC and insulin-like growth factor [IGF]), and signatures related to poor clinical outcome. In the classical type, there was a significant correlation in the copy number variation of the iCCA and HCC components, suggesting a clonal origin. Exome sequencing revealed an average of 63 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (6/21, 29%) in HCC-CCAs.Conclusions: Mixed HCC-CCA represents a heterogeneous group of tumors, with the stem-cell type characterized by features of poor prognosis, and the classical type with common lineage for HCC and iCCA components. CLC stands alone as a distinct biliary-derived entity associated with chromosomal stability and active TGF-b signaling.Lay summary: Molecular analysis of mixed hepatocellular cholangiocarcinoma (HCC-CCA) showed that cholangiolocellular carcinoma (CLC) is distinct and biliary in origin. It has none of the traits of hepatocellular carcinoma (HCC). However, within mixed HCC-CCA, stem-cell type tumors shared an aggressive nature and poor outcome, whereas the classic type showed a common cell lineage for both the HCC and the intrahepatic CCA component. The pathological classification of mixed HCC-CCA should be redefined because of the new molecular data provided. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

Co-expression gene network analysis reveals novel regulatory pathways involved in porto-sinusoidal vascular disease

[Background & Aims] Porto-sinusoidal vascular disease (PSVD) is a rare vascular liver disease of unknown etiology that causes portal hypertension. It usually affects young individuals and shortens live expectancy. The deregulated pathways involved in PSVD development are unknown and therefore we lack curative treatments. The purpose of this study was to integrate transcriptomic and clinical data by comprehensive network-based modeling in order to uncover altered biological processes in patients with PSVD.[Methods] We obtained liver tissue samples from 20 consecutive patients with PSVD and 21 sex- and age-matched patients with cirrhosis and 13 histologically normal livers (HNL) (initial cohort) and performed transcriptomic analysis. Microarray data were analyzed using weighted gene correlation network analysis to identify clusters of highly correlated genes differently expressed in patients with PSVD. We next evaluated the molecular pathways enriched in patients with PSVD and the core-related genes from the most significantly enriched pathways in patients with PSVD. Our main findings were validated using RNA sequencing in a different cohort of PSVD, cirrhosis and HNL (n = 8 for each group).[Results] Patients with PSVD have a distinctive genetic profile enriched mainly in canonical pathways involving hemostasis and coagulation but also lipid metabolism and oxidative phosphorylation. Serpin family (SERPINC1), the apolipoproteins (APOA, APOB, APOC), ATP synthases (ATP5G1, ATP5B), fibrinogen genes (FGB, FGA) and alpha-2-macroglobulin were identified as highly connective genes that may have an important role in PSVD pathogenesis.[Conclusion] PSVD has a unique transcriptomic profile and we have identified deregulation of pathways involved in vascular homeostasis as the main pathogenic event of disease development. [Lay summary] Porto-sinusoidal vascular disease is a rare but life-shortening disease that affects mainly young people. Knowledge of the disrupted pathways involved in its development will help to identify novel therapeutic targets and new treatments. Using a systems biology approach, we identify that pathways regulating endothelial function and tone may act as drivers of porto-sinusoidal vascular disease.This study was supported by the Instituto de Salud Carlos III FIS PI17/00398, the Ministry of Education and Science, Spain (SAF-2016-75767-R); Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR-SGR2017-517) a grant from Generalitat de Catalunya, Fondo Europeo de Desarrollo Regional (FEDER) and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), funded by Instituto de Salud Carlos III. Marta Magaz is a recipient of a Río Hortega grant from Instituto de Salud Carlos III. Pol Olivas has been funded by Contractes Clínic de Recerca ”Emili Letang-Josep Font’’ 2020, granted by Hospital Clínic de Barcelona.Peer reviewe

Els receptors ErbB en la resposta a l’estimulació adrenèrgica crònica en el ratolí

[cat] En aquest estudi s’ha abordat el paper que poden jugar els receptors de la família ErbB en l’estrès crònic per catecolamines en el ratolí. Se sabia que el Factor de Creixement Epidèrmic (EGF) interfereix en el fetge, el teixit adipós i el cor en la resposta màxima d’aquests òrgans a l’adrenalina disminuint els nivells de cAMP. A més a més, l’EGF és alliberat de les glàndules submaxil•lars en resposta a un estímul α-adrenèrgic. Aquesta interferència en el cor té un efecte cardioprotector durant una estimulació adrenèrgica aguda. En models fisiològics d’estrès per confrontació entre mascles, es va observar que després de d’un període de lluita els nivells d’EGF en sang augmentaven fins a 1000 vegades durant la primera hora. En aquests animals el cor presentava un aspecte normal. En animals en els que se’ls havia extirpat les glàndules submaxil•lars (SIALO), després d’un període de lluita presentaven disfuncions cardíaques tant morfològiques com funcionals. Vist el paper protector agut de l’EGF, el nostre primer objectiu va ser estudiar l’efecte que l’EGF procedent de les glàndules submaxil•lars pot jugar en l’estimulació adrenèrgica crònica i en els processos d’hipertròfia cardíaca derivats d’aquest, ja que se sap que el receptor ErbB1 hi està involucrat. Els resultats han demostrat que dos dies d’estimulació adrenèrgica buiden les glàndules submaxil•lars del contingut d’EGF i no deixa suficient temps per a que es reomplin. Un nou estímul adrenèrgic després de dos dies de tractament amb catecolamines no provoca cap augment d’EGF en plasma. A més a més, després de 7 dies de tractament amb isoproterenol (un agonista β-adrenèrgic) els animals SIALO presenten els mateixos valors que característicament defineixen la hipertròfia cardíaca que els animals control, així com també els mateixos nivells dels receptors ErBB1, ErbB2 i ErbB4. Aquests resultats, doncs, ens porten a concloure que l’EGF procedent de les glàndules submaxil•lars no contribueix directament en els processos derivats de l’estimulació adrenèrgica crònica. Observacions realitzades en aquests estudis van derivar en altres línies d’investigació. Així, la segona part d’aquesta tesi està dedicada a la caracterització del receptor ErbB3 en cardiomiòcits de ratolí. A finals dels anys 90 es va descriure que els receptors ErbB presents en el cor adult eren només ErbB1, ErbB2 i ErbB4. En aquest segon estudi nosaltres demostrem la presència d’erbB3 en el cor de ratolí des del naixement fina a la vida adulta. També mostrem la seva localització en els túbuls T de les línies Z, així com la seva activació quan el cor és estimulat amb Neuregulina-1β, un dels seus lligands. Finalment, també mostrem la seva regulació a través de l’estimulació adrenèrgica. En l’últim apartat, el nostre centre d’atenció es troba en el ronyó. Nosaltres descrivim que després de 7 dies de tractament amb isoproterenol observem una disminució reversible en el pes dels ronyons tractats, així com un menor contingut de proteïna renal. Paral•lelament, observem un augment de la proliferació cel•lular en els túbuls de les nefrones, especialment en la zona interior del còrtex. A més a més, també observem un increment reversible de l’expressió dels quatre receptors ErbB. No obstant, no observem disfunció renal. Hem observat també que els quatre receptors es localitzen en les mateixes zones de la nefrona, i que interaccionen entre ells. Tots aquests fets ens han portat a hipotetitzar que el tractament crònic amb isoproterenol causa una lesió lleu en els túbuls renals i que els sistema de receptors ErbB està implicat en els subseqüents processos de regeneració que s’observen després d’un estímul perjudicial.[eng] In this thesis, we have studied the role of ErbB receptors during catecholamine-induced stress in mice. This study has been divided in three major areas. Our first objective was to study the effect that Submandibular Glands (SMG) Epidermal Growth Factor (EGF) may play in the heart during chronic adrenergic stimulation and in the subsequent hypertrophy processes. Our results show how 2 days of adrenergic treatment empty the EGF content in SMG, also suggesting a low-rate of new EGF synthesis in SMG. A new adrenergic stimulus after two days of treatment does not increase the EGF plasma concentration anymore. In addition, after 7 days of treatment with isoproterenol, sialoadenectomized animals present the same characteristics as control animals when analyzing heart hypertrophy, as well as the same levels of all ErbB receptors. These results suggest that SMG’s EGF does neither directly contribute nor interferes in the processes derived from chronic adrenergic stimulation such as heart hypertrophy. The second chapter of this thesis was focused in the characterization of ErbB3 receptor in mice adult heart cardiomyocytes. We demonstrated the presence of ErbB3 receptor in mouse heart since birth until adulthood. We also showed its localization in the T-tubules in the Z-lines, as well as its activation upon Neuregulin-1β stimulation. Finally, we also showed its regulation by adrenergic stimulation in adult mice hearts. In the last chapter, we studied the ErbB receptors in the kidney during chronic adrenergic treatment. We observed a reversible weight and protein content loss after 7 days of adrenergic treatment. We also observed an increase in cellular proliferation in the kidney tubules, mainly in the inner cortex area, which coincided with an increase in the expression of all ErbB receptors in the kidney. However, we did not observe any renal dysfunction. We also showed that all four ErbB receptors were expressed in the same areas in the kidney and interacted with each other. All these results made us hypothesize that chronic adrenergic treatment may cause a mild injury in renal tubules. We think ErbB receptors may be involved in the subsequent regeneration processes observed after this injury stimulus

The Pathophysiology of Portal Vein Thrombosis in Cirrhosis: Getting Deeper into Virchows Triad

Portal vein thrombosis (PVT) is a common complication among patients with cirrhosis. However, its pathophysiology is not well established and there are currently very few predictive factors, none of which are actually useful, from a clinical perspective. The contribution of each of the vertices of Virchow's triad, e.g., blood hypercoagulability, blood flow, and portal vein endothelial damage in the development of PVT is not clear. In this review, we aim to recapitulate the latest studies on the field of PVT development in order to understand its mechanisms and discuss some of the future directions in the study of this important complication of cirrhosis

Impaired endothelial autophagy promotes liver fibrosis by aggravating the oxidative stress response during acute liver injury.

BACKGROUND & AIMS Endothelial dysfunction plays an essential role in liver injury, yet the phenotypic regulation of liver sinusoidal endothelial cells (LSECs) remains unknown. Autophagy is an endogenous protective system whose loss could undermine LSEC integrity and phenotype. The aim of our study was to investigate the role of autophagy in the regulation of endothelial dysfunction and the impact of its manipulation during liver injury. METHODS We analyzed primary isolated LSECs from Atg7 and Atg7 mice as well as rats after CCl induced liver injury. Liver tissue and primary isolated stellate cells were used to analyze liver fibrosis. Autophagy flux, microvascular function, nitric oxide bioavailability, cellular superoxide content and the antioxidant response were evaluated in endothelial cells. RESULTS Autophagy maintains LSEC homeostasis and is rapidly upregulated during capillarization in vitro and in vivo. Pharmacological and genetic downregulation of endothelial autophagy increases oxidative stress in vitro. During liver injury in vivo, the selective loss of endothelial autophagy leads to cellular dysfunction and reduced intrahepatic nitric oxide. The loss of autophagy also impairs LSECs ability to handle oxidative stress and aggravates fibrosis. CONCLUSIONS Autophagy contributes to maintaining endothelial phenotype and protecting LSECs from oxidative stress during early phases of liver disease. Selectively potentiating autophagy in LSECs during early stages of liver disease may be an attractive approach to modify the disease course and prevent fibrosis progression. LAY SUMMARY Liver endothelial cells are the first liver cell type affected after any kind of liver injury. The loss of their unique phenotype during injury amplifies liver damage by orchestrating the response of the liver microenvironment. Autophagy is a mechanism involved in the regulation of this initial response and its manipulation can modify the progression of liver damage

Spermidine Supplementation Protects the Liver Endothelium from Liver Damage in Mice

Chronic liver diseases are multifactorial and the need to develop effective therapies is high. Recent studies have shown the potential of ameliorating liver disease progression through protection of the liver endothelium. Polyamine spermidine (SPD) is a caloric restriction mimetic with autophagy-enhancing properties capable of prolonging lifespan and with a proven beneficial effect in cardiovascular disease in mice and humans. We evaluated the use of dietary supplementation with SPD in two models of liver disease (CCl4 and CDAAH diet). We analyzed the effect of SPD on endothelial dysfunction in vitro and in vivo. C57BL/6J mice were supplemented with SPD in the drinking water prior and concomitantly with CCl4 and CDAAH treatments. Endothelial autophagy deficient (Atg7endo) mice were also evaluated. Liver tissue was used to evaluate the impact of SPD prophylaxis on liver damage, endothelial dysfunction, oxidative stress, mitochondrial status, inflammation and liver fibrosis. SPD improved the endothelial response to oxidative injury in vitro and improved the liver endothelial phenotype and protected against liver injury in vivo. SPD reduced the overall liver oxidative stress and improved mitochondrial fitness. The absence of benefits in the Atg7endo mice suggests an autophagy-dependent effect of SPD. This study suggests SPD diet supplementation in early phases of disease protects the liver endothelium from oxidative stress and may be an attractive approach to modify the chronic liver disease course and halt fibrosis progression

Mixed hepatocellular cholangiocarcinoma tumors: Cholangiolocellular carcinoma is a distinct molecular entity

Background & Aims: Mixed hepatocellular cholangiocarcinoma (HCC-CCA) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy.Methods: Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 18 patients with mixed HCC-CCA were performed, encompassing the whole histological spectrum of the disease. Comparative genomic analysis was carried out, using independent datasets of HCC (n = 164) and intrahepatic cholangiocarcinoma (iCCA) (n = 149).Results: Integrative genomic analysis of HCC-CCAs revealed that cholangiolocellular carcinoma (CLC) represents a distinct biliaryderived entity compared with the stem-cell and classical types. CLC tumors were neural cell adhesion molecule (NCAM) positive (6/6 vs. 1/12, p < 0.001), chromosomally stable (mean chromosomal aberrations 5.7 vs. 14.1, p = 0.008), showed significant upregulation of transforming growth factor (TGF)-beta signaling and enrichment of inflammation-related and immune response signatures (p < 0.001). Stem-cell tumors were characterized by spaltlike transcription factor 4 (SALL4) positivity (6/8 vs. 0/10, p < 0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., MYC and insulin-like growth factor [IGF]), and signatures related to poor clinical outcome. In the classical type, there was a significant correlation in the copy number variation of the iCCA and HCC components, suggesting a clonal origin. Exome sequencing revealed an average of 63 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (6/21, 29%) in HCC-CCAs.Conclusions: Mixed HCC-CCA represents a heterogeneous group of tumors, with the stem-cell type characterized by features of poor prognosis, and the classical type with common lineage for HCC and iCCA components. CLC stands alone as a distinct biliary-derived entity associated with chromosomal stability and active TGF-b signaling.Lay summary: Molecular analysis of mixed hepatocellular cholangiocarcinoma (HCC-CCA) showed that cholangiolocellular carcinoma (CLC) is distinct and biliary in origin. It has none of the traits of hepatocellular carcinoma (HCC). However, within mixed HCC-CCA, stem-cell type tumors shared an aggressive nature and poor outcome, whereas the classic type showed a common cell lineage for both the HCC and the intrahepatic CCA component. The pathological classification of mixed HCC-CCA should be redefined because of the new molecular data provided. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved