Publications by authors named "Femke Heindryckx"

30 Publications

  • Page 1 of 1

Anthracyclins Increase PUFAs: Potential Implications in ER Stress and Cell Death.

Cells 2021 May 11;10(5). Epub 2021 May 11.

Analytical Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden.

Metabolic and personalized interventions in cancer treatment require a better understanding of the relationship between the induction of cell death and metabolism. Consequently, we treated three primary liver cancer cell lines with two anthracyclins (doxorubicin and idarubin) and studied the changes in the lipidome. We found that both anthracyclins in the three cell lines increased the levels of polyunsaturated fatty acids (PUFAs) and alkylacylglycerophosphoethanolamines (etherPEs) with PUFAs. As PUFAs and alkylacylglycerophospholipids with PUFAs are fundamental in lipid peroxidation during ferroptotic cell death, our results suggest supplementation with PUFAs and/or etherPEs with PUFAs as a potential general adjuvant of anthracyclins. In contrast, neither the markers of de novo lipogenesis nor cholesterol lipids presented the same trend in all cell lines and treatments. In agreement with previous research, this suggests that modulation of the metabolism of cholesterol could be considered a specific adjuvant of anthracyclins depending on the type of tumor and the individual. Finally, in agreement with previous research, we found a relationship across the different cell types between: (i) the change in endoplasmic reticulum (ER) stress, and (ii) the imbalance between PUFAs and cholesterol and saturated lipids. In the light of previous research, this imbalance partially explains the sensitivity to anthracyclins of the different cells. In conclusion, our results suggest that the modulation of different lipid metabolic pathways may be considered for generalized and personalized metabochemotherapies.
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http://dx.doi.org/10.3390/cells10051163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151859PMC
May 2021

Activated platelets contribute to the progression of hepatocellular carcinoma by altering the tumor environment.

Life Sci 2021 May 12;277:119612. Epub 2021 May 12.

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. Electronic address:

Aim: Hepatocellular carcinoma (HCC) is a primary liver cancer that usually develops in a background of chronic liver disease and prolonged inflammation. A major contributor in the complex molecular pathogenesis of HCC is the highly intertwined cross-talk between the tumor and the surrounding stromal cells, such as hepatic stellate cells, endothelial cells, macrophages and other immune cells. These tumor-stroma interactions actively fuel tumor growth and modulate the hepatic microenvironment to benefit tumor invasion and disease progression. Platelets have been reported to interact with different cell types in the tumor microenvironment, including tumor cells, stellate cells and macrophages.

Materials And Methods: Mice were treated with hepatocarcinogenic compound diethylnitrosamine for 25 weeks to induce HCC in the background of fibrosis and inflammation. From week 10, anti-platelet drug Clopidogrel was added to the drinking water and mice were given ad libitum access.

Key Findings: In this study, we show that activated platelets promote tumor cell proliferation and contribute to the adverse tumor-stroma cross-talk that fuels tumor progression. We also show that inhibiting platelet activation with the P2Y12-inhibitor Clopidogrel decreases the number of tumors in a chemically induced mouse model for HCC.

Significance: These results suggest an important role for platelets in the pathogenesis of HCC and that the use of anti-platelet drugs may be therapeutically relevant for patients with liver cancer.
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http://dx.doi.org/10.1016/j.lfs.2021.119612DOI Listing
May 2021

Inhibiting P2Y12 in Macrophages Induces Endoplasmic Reticulum Stress and Promotes an Anti-Tumoral Phenotype.

Int J Mol Sci 2020 Oct 31;21(21). Epub 2020 Oct 31.

Medical Cell Biology, Uppsala University, 75123 Uppsala, Sweden.

The P2Y12 receptor is an adenosine diphosphate responsive G protein-coupled receptor expressed on the surface of platelets and is the pharmacologic target of several anti-thrombotic agents. In this study, we use liver samples from mice with cirrhosis and hepatocellular carcinoma to show that P2Y12 is expressed by macrophages in the liver. Using in vitro methods, we show that inhibition of P2Y12 with ticagrelor enhances tumor cell phagocytosis by macrophages and induces an anti-tumoral phenotype. Treatment with ticagrelor also increases the expression of several actors of the endoplasmic reticulum (ER) stress pathways, suggesting activation of the unfolded protein response (UPR). Inhibiting the UPR with tauroursodeoxycholic acid (Tudca) diminishes the pro-phagocytotic effect of ticagrelor, thereby indicating that P2Y12 mediates macrophage function through activation of ER stress pathways. This could be relevant in the pathogenesis of chronic liver disease and cancer, as macrophages are considered key players in these inflammation-driven pathologies.
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http://dx.doi.org/10.3390/ijms21218177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672568PMC
October 2020

Inhibiting IRE1α-endonuclease activity decreases tumor burden in a mouse model for hepatocellular carcinoma.

Elife 2020 10 26;9. Epub 2020 Oct 26.

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.

Hepatocellular carcinoma (HCC) is a liver tumor that usually arises in patients with cirrhosis. Hepatic stellate cells are key players in the progression of HCC, as they create a fibrotic micro-environment and produce growth factors and cytokines that enhance tumor cell proliferation and migration. We assessed the role of endoplasmic reticulum (ER) stress in the cross-talk between stellate cells and HCC cells. Mice with a fibrotic HCC were treated with the IRE1α-inhibitor 4μ8C, which reduced tumor burden and collagen deposition. By co-culturing HCC-cells with stellate cells, we found that HCC-cells activate IREα in stellate cells, thereby contributing to their activation. Inhibiting IRE1α blocked stellate cell activation, which then decreased proliferation and migration of tumor cells in different in vitro 2D and 3D co-cultures. In addition, we also observed cell-line-specific direct effects of inhibiting IRE1α in tumor cells.
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http://dx.doi.org/10.7554/eLife.55865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661042PMC
October 2020

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties.

J Vis Exp 2020 08 7(162). Epub 2020 Aug 7.

Department of Medical Cell Biology, Uppsala University;

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the in vivo situation compared to traditional 2D cultures. Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.
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http://dx.doi.org/10.3791/61606DOI Listing
August 2020

Fibrin fragment E potentiates TGF-β-induced myofibroblast activation and recruitment.

Cell Signal 2020 08 22;72:109661. Epub 2020 Apr 22.

Dept. of Medical Cell Biology, Uppsala University, P.O. Box 571, SE-751 23 Uppsala, Sweden; Dept. of Radiology, Uppsala University Hospital, SE-751 85 Uppsala, Sweden.

Fibrin is an essential constituent of the coagulation cascade, and the formation of hemostatic fibrin clots is central to wound healing. Fibrin clots are over time degraded into fibrin degradation products as the injured tissue is replaced by granulation tissue. Our goal was to study the role of the fibrin degradation product fragment E (FnE) in fibroblast activation and migration. We present evidence that FnE is a chemoattractant for fibroblasts and that FnE can potentiate TGF-β-induced myofibroblast formation. FnE forms a stable complex with αβ integrin, and the integrin β subunit is required both for FnE-induced fibroblast migration and for potentiation of TGF-β-induced myofibroblast formation. Finally, subcutaneous infusion of FnE in mice results in a fibrotic response in the hypodermis. These results support a model where FnE released from clots in wounded tissue promote wound healing and fibrosis by both recruitment and activation of fibroblasts. Fibrin fragment E could thus represent a therapeutic target for treatment of pathological fibrosis.
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http://dx.doi.org/10.1016/j.cellsig.2020.109661DOI Listing
August 2020

Liver Cancer Cell Lines Treated with Doxorubicin under Normoxia and Hypoxia: Cell Viability and Oncologic Protein Profile.

Cancers (Basel) 2019 Jul 20;11(7). Epub 2019 Jul 20.

Department of Pharmacy, Uppsala University, Box 580, 751 23 Uppsala, Sweden.

Hepatocellular carcinoma is often treated with a combination of doxorubicin and embolization, exposing it to high concentrations and hypoxia. Separation of the possible synergistic effect of this combination in vivo is difficult. Here, treatment with doxorubicin, under hypoxia or normoxia in different liver cancer cell lines, was evaluated. Liver cancer cells HepG2, Huh7, and SNU449 were exposed to doxorubicin, hypoxia, or doxorubicin + hypoxia with different duration. Treatment response was evaluated with cell viability, apoptosis, oxidative stress, and summarized with IC. The protein profile of a 92-biomarker panel was analyzed on cells treated with 0 or 0.1 µM doxorubicin during 6 or 72 h, under normoxia or hypoxia. Hypoxia decreased viability of HepG2 and SNU499. HepG2 was least and SNU449 most tolerant to doxorubicin treatment. Cytotoxicity of doxorubicin increased over time in HepG2 and Huh7. The combination of doxorubicin + hypoxia affected the cells differently. Normalized protein expression was lower for HepG2 than Huh7 and SNU449. Hierarchical clustering separated HepG2 from Huh7 and SNU449. These three commonly used cell lines have critically different responses to chemotherapy and hypoxia, which was reflected in their different protein expression profile. These different responses suggest that tumors can respond differently to the combination of local chemotherapy and embolization.
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http://dx.doi.org/10.3390/cancers11071024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678640PMC
July 2019

Platelets as Key Factors in Hepatocellular Carcinoma.

Cancers (Basel) 2019 Jul 20;11(7). Epub 2019 Jul 20.

Department of Medical Cell Biology, Uppsala University, Box 571, Husargatan 3, 75-431 Uppsala, Sweden.

Hepatocellular carcinoma (HCC) is a primary liver cancer that usually develops in the setting of chronic inflammation and liver damage. The hepatic microenvironment plays a crucial role in the disease development, as players such as hepatic stellate cells, resident liver macrophages (Kupffer cells), endothelial cells, extracellular matrix, and a variety of immune cells interact in highly complex and intertwined signaling pathways. A key factor in these cross-talks are platelets, whose role in cancer has gained growing evidence in recent years. Platelets have been reported to promote HCC cell proliferation and invasion, but their involvement goes beyond the direct effect on tumor cells, as they are known to play a role in pro-fibrinogenic signaling and the hepatic immune response, as well as in mediating interactions between these factors in the stroma. Anti-platelet therapy has been shown to ameliorate liver injury and improve the disease outcome. However, platelets have also been shown to play a crucial role in liver regeneration after organ damage. Therefore, the timing and microenvironmental setting need to be kept in mind when assessing the potential effect and therapeutic value of platelets in the disease progression, while further studies are needed for understanding the role of platelets in patients with HCC.
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http://dx.doi.org/10.3390/cancers11071022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678690PMC
July 2019

Role of proteoglycans in neuro-inflammation and central nervous system fibrosis.

Matrix Biol 2018 08 31;68-69:589-601. Epub 2018 Jan 31.

Department of Medical Biochemistry and Microbiology/SciLifeLab, Uppsala University, Uppsala, Sweden. Electronic address:

Fibrosis is defined as the thickening and scarring of connective tissue, usually as a consequence of tissue damage. The central nervous system (CNS) is special in the sense that fibrogenic cells are restricted to vascular and meningeal areas. Inflammation and the disruption of the blood-brain barrier can lead to the infiltration of fibroblasts and trigger fibrotic response. While the initial function of the fibrotic tissue is to restore the blood-brain barrier and to limit the site of injury, it also demolishes the structure of extracellular matrix and impedes the healing process by producing inhibitory molecules and forming a physical and biochemical barrier that prevents axon regeneration. As a major constituent in the extracellular matrix, proteoglycans participate in the neuro-inflammation, modulating the fibrotic process. In this review, we will discuss the pathophysiology of fibrosis during acute injuries of the CNS, as well as during chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and age-related neurodegeneration with focus on the functional roles of proteoglycans.
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http://dx.doi.org/10.1016/j.matbio.2018.01.015DOI Listing
August 2018

Macrophage Depletion Attenuates Extracellular Matrix Deposition and Ductular Reaction in a Mouse Model of Chronic Cholangiopathies.

PLoS One 2016 12;11(9):e0162286. Epub 2016 Sep 12.

Liver Cell Biology Laboratory, Department of Basic Biomedical Sciences, Vrije Universiteit Brussel (VUB), Brussel, Belgium.

Chronic cholangiopathies, such as primary and secondary sclerosing cholangitis, are progressive disease entities, associated with periportal accumulation of inflammatory cells, encompassing monocytes and macrophages, peribiliary extracellular matrix (ECM) deposition and ductular reaction (DR). This study aimed to elucidate the relevance of macrophages in the progression of chronic cholangiopathies through macrophage depletion in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mouse model. One group of mice received a single i.p. injection of Clodronate encapsulated liposomes (CLOLipo) at day 7 of a 14 day DDC treatment, while control animals were co-treated with PBSLipo instead. Mice were sacrificed after 7 or respectively 14 days of treatment for immunohistochemical assessment of macrophage recruitment (F4/80), ECM deposition (Sirius Red, Laminin) and DR (CK19). Macrophage depletion during a 14 day DDC treatment resulted in a significant inhibition of ECM deposition. Porto-lobular migration patterns of laminin-rich ECM and ductular structures were significantly attenuated and a progression of DR was effectively inhibited by macrophage depletion. CLOLipo co-treatment resulted in a confined DR to portal regions without amorphous cell clusters. This study suggests that therapeutic options selectively directed towards macrophages might represent a feasible treatment for chronic cholestatic liver diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162286PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5019458PMC
August 2017

Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing.

EMBO Mol Med 2016 07 1;8(7):729-44. Epub 2016 Jul 1.

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden Department of Radiology, Uppsala University Hospital, Uppsala, Sweden

ER stress results in activation of the unfolded protein response and has been implicated in the development of fibrotic diseases. In this study, we show that inhibition of the ER stress-induced IRE1α signaling pathway, using the inhibitor 4μ8C, blocks TGFβ-induced activation of myofibroblasts in vitro, reduces liver and skin fibrosis in vivo, and reverts the fibrotic phenotype of activated myofibroblasts isolated from patients with systemic sclerosis. By using IRE1α(-/-) fibroblasts and expression of IRE1α-mutant proteins lacking endoribonuclease activity, we confirmed that IRE1α plays an important role during myofibroblast activation. IRE1α was shown to cleave miR-150 and thereby to release the suppressive effect that miR-150 exerted on αSMA expression through c-Myb. Inhibition of IRE1α was also demonstrated to block ER expansion through an XBP-1-dependent pathway. Taken together, our results suggest that ER stress could be an important and conserved mechanism in the pathogenesis of fibrosis and that components of the ER stress pathway may be therapeutically relevant for treating patients with fibrotic diseases.
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http://dx.doi.org/10.15252/emmm.201505925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931288PMC
July 2016

Time-dependent effect of hypoxia on tumor progression and liver progenitor cell markers in primary liver tumors.

PLoS One 2015 20;10(3):e0119555. Epub 2015 Mar 20.

Gastro-enterology and hepatology, Ghent University, Ghent, Belgium.

Background & Aims: Expression of liver progenitor cell (LPC) characteristics has been proposed as a negative prognostic marker in primary liver tumors. Hypoxia has been linked to activation of the Notch pathway which is responsible for activation and proliferation of LPCs and hypoxia-induced LPC activation has been shown in hepatocellular carcinoma. Our aim was to elucidate the time-dependent effects of hypoxia on the LPC niche in hepatocellular carcinoma which could aid in determining a safe time frame for use of hypoxia inducing therapies.

Methods: We used dimethyloxaloylglycine to mimic a hypoxic reaction in mice by stabilizing hypoxia-inducible factor 1 alpha at three distinct time points in diethylnitrosamine induced hepatocarcinogenesis. LPC, metastasis and Notch pathway markers were determined by quantitative PCR and (immune)histochemistry (heamatoxillin-eosin, reticulin, Sirius red and cytokeratin 19 staining).

Results: Activating the hypoxia inducible pathway early in hepatocarcinogenesis resulted in an increased incidence of both cholangioma and hepatocellular lesions, associated with high expression of LPC, metastatic and Notch pathway markers. Adversely, activating the hypoxic response during tumor development resulted in decreased incidence of hepatocellular lesions and increased cholangioma incidence, with an unaltered gene expression profile of LPC-, Notch pathway- and metastatic markers. A hypoxic insult at advanced stages of hepatocarcinogenesis severely increased the expression of LPC characteristics, however without increased expression of actors of the Notch pathway and metastatic markers and minor changes in incidence of hepatocellular and cholangioma lesions.

Conclusion: Our results indicate that increased hypoxia at the onset of tumor development has detrimental effects on tumor progression; patients with HCC developed in a background of fibrosis/cirrhosis might therefore represent a more difficult treatment group. In contrast, hypoxia during tumor development appears to favor tumor outcome, highlighting the importance of early detection. Finally, hypoxia in advanced stages resulted in increased expression of LPC characteristics indicating poor outcome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119555PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368520PMC
February 2016

Targeting the tumor stroma in hepatocellular carcinoma.

World J Hepatol 2015 Feb;7(2):165-76

Femke Heindryckx, Pär Gerwins, Department of Medical Biochemistry and Microbiology, Biomedical Research Center, Uppsala University, SE-75123 Uppsala, Sweden.

Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers worldwide. In ninety percent of the cases it develops as a result of chronic liver damage and it is thus a typical inflammation-related cancer characterized by the close relation between the tumor microenvironment and tumor cells. The stromal environment consists out of several cell types, including hepatic stellate cells, macrophages and endothelial cells. They are not just active bystanders in the pathogenesis of HCC, but play an important and active role in tumor initiation, progression and metastasis. Furthermore, the tumor itself influences these cells to create a background that is beneficial for sustaining tumor growth. One of the key players is the hepatic stellate cell, which is activated during liver damage and differentiates towards a myofibroblast-like cell. Activated stellate cells are responsible for the deposition of extracellular matrix, increase the production of angiogenic factors and stimulate the recruitment of macrophages. The increase of angiogenic factors (which are secreted by macrophages, tumor cells and activated stellate cells) will induce the formation of new blood vessels, thereby supplying the tumor with more oxygen and nutrients, thus supporting tumor growth and offering a passageway in the circulatory system. In addition, the secretion of chemokines by the tumor cells leads to the recruitment of tumor associated macrophages. These tumor associated macrophages are key actors of cancer-related inflammation, being the main type of inflammatory cells infiltrating the tumor environment and exerting a tumor promoting effect by secreting growth factors, stimulating angiogenesis and influencing the activation of stellate cells. This complex interplay between the several cell types involved in liver cancer emphasizes the need for targeting the tumor stroma in HCC patients.
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http://dx.doi.org/10.4254/wjh.v7.i2.165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342599PMC
February 2015

Hepatitis mouse models: from acute-to-chronic autoimmune hepatitis.

Int J Exp Pathol 2014 Oct 12;95(5):309-20. Epub 2014 Aug 12.

Department of Hepatology and Gastroenterology, Ghent University, Ghent, Belgium; Department of Endocrinology, Yale School of Medicine, New Haven, CT, USA.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease associated with interface hepatitis, raised plasma liver enzymes, the presence of autoantibodies and regulatory T-cell (Tregs) dysfunction. The clinical course is heterogeneous, manifested by a fulminant or indolent course. Although genetic predisposition is well accepted, the combination with currently undefined environmental factors is crucial for the development of the disease. Progress in the development of reliable animal models provides added understanding of the pathophysiology of AIH, and these will be very useful in evaluating potential therapeutics. It appears that artificially breaking tolerance in the liver is easy. However, maintaining this state of tolerance breakdown, to get chronic hepatitis, is difficult because liver immune homeostasis is strongly regulated by several immune response inhibitory mechanisms. For example, Tregs are crucial regulators in acute and chronic hepatitis, and C57BL/6 mice are most prone to experimental AIH. Immunization of C57BL/6 mice with liver (AIH) autoantigens (CYP2D6/FTCD or IL-4R) and the disturbance of liver regulatory mechanism(s), leading to experimental AIH, are likely to be most representative of human AIH pathology.
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http://dx.doi.org/10.1111/iep.12090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4209923PMC
October 2014

The roles of transforming growth factor-β, Wnt, Notch and hypoxia on liver progenitor cells in primary liver tumours (Review).

Int J Oncol 2014 Apr 3;44(4):1015-22. Epub 2014 Feb 3.

Department of Gastroenterology and Hepatology, 1K12, Ghent University Hospital, 9000 Gent, Belgium.

Primary liver tumours have a high incidence and mortality. The most important forms are hepatocellular carcinoma and intrahepatic cholangiocarcinoma, both can occur together in the mixed phenotype hepatocellular-cholangiocarcinoma. Liver progenitor cells (LPCs) are bipotential stem cells activated in case of severe liver damage and are capable of forming both cholangiocytes and hepatocytes. Possibly, alterations in Wnt, transforming growth factor-β, Notch and hypoxia pathways in these LPCs can cause them to give rise to cancer stem cells, capable of driving tumourigenesis. In this review, we summarize and discuss current knowledge on the role of these pathways in LPC activation and differentiation during hepatocarcinogenesis.
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http://dx.doi.org/10.3892/ijo.2014.2286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977811PMC
April 2014

The paradox of the unfolded protein response in cancer.

Anticancer Res 2013 Nov;33(11):4683-94

Department of Hepatology and Gastroenterology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium.

The endoplasmic reticulum (ER) is an elaborate organelle that is essential for cellular function and survival. Conditions that interfere with ER functioning can lead to the accumulation of unfolded proteins, which are detected by transmembrane sensors that then initiate the unfolded protein response (UPR) to restore ER proteostasis. If the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, particularly because ER stress-induced apoptosis is implicated in the pathophysiology of several diseases, including cancer. Whether the UPR inhibits tumour growth or protects tumour cells by facilitating their adaptation to stressful conditions within the tumour microenvironment is unknown, and dissection of the UPR network will likely provide answers to this question. In this review, we aim to elucidate the paradoxical role of the UPR in apoptosis and cancer.
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November 2013

The need for transparency and good practices in the qPCR literature.

Nat Methods 2013 Nov;10(11):1063-7

Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford, UK.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
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http://dx.doi.org/10.1038/nmeth.2697DOI Listing
November 2013

MicroRNA-24 suppression of N-deacetylase/N-sulfotransferase-1 (NDST1) reduces endothelial cell responsiveness to vascular endothelial growth factor A (VEGFA).

J Biol Chem 2013 Sep 24;288(36):25956-25963. Epub 2013 Jul 24.

From the Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Husargatan 3, P. O. Box 582, SE-751 23 Uppsala,. Electronic address:

Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.
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http://dx.doi.org/10.1074/jbc.M113.484360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764800PMC
September 2013

Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.

Hepatology 2013 May 14;57(5):1793-805. Epub 2013 Mar 14.

Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium.

Unlabelled: The pathophysiology of nonalcoholic steatohepatitis (NASH) should be approached as a multifactorial process. In several stages of NASH, a link between disease progression and hepatic microvasculature changes can be made. In this study we investigated the role of angiogenesis in two mouse models for NASH, and the effect of a preventive and therapeutic antiangiogenic treatment in a diet-induced mouse model for NASH. Protein and RNA levels of angiogenic and inflammatory factors were significantly up-regulated in the liver of C56BL/6 and db/db mice with NASH at different timepoints. To examine the effect of angiogenic factors on the disease progression of NASH, a prevention and treatment study was set up, blocking the placental growth factor (PlGF) or vascular endothelial growth factor receptor 2 (VEGFR2). Our study showed that treatment prevents the progression of NASH by attenuating steatosis and inflammation, both in a preventive and therapeutic setting, thereby confirming the hypothesis that angiogenic factors play an early role in the disease progression from steatosis to NASH. Anti-PlGF (αPlGF) did not significantly improve liver histology. Vascular corrosion casting showed a more disrupted liver vasculature in mice with NASH compared to controls. Treatment with αVEGFR2 showed an improvement of the liver vasculature. Moreover, fat-laden primary hepatocytes treated with αVEGFR2 stored significantly less lipids.

Conclusion: Our results demonstrate that there is an increased expression of angiogenic factors in the liver in different mouse models for NASH. We found that VEGFR2 blockage attenuates steatosis and inflammation in a diet-induced mouse model for NASH in a preventive and therapeutic setting. Our findings warrant further investigation of the role of angiogenesis in the pathophysiology in NASH.
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http://dx.doi.org/10.1002/hep.26219DOI Listing
May 2013

The placental growth factor as a target against hepatocellular carcinoma in a diethylnitrosamine-induced mouse model.

J Hepatol 2013 Feb 6;58(2):319-28. Epub 2012 Oct 6.

Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium.

Background & Aims: The placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family known to stimulate endothelial cell growth, migration and survival, attract angiocompetent macrophages, and determine the metastatic niche. Unlike VEGF, genetic studies have shown that PlGF is specifically involved in pathologic angiogenesis, thus its inhibition would not affect healthy blood vessels, providing an attractive drug candidate with a good safety profile.

Methods: We assess whether inhibition of PlGF could be used as a potential therapy against hepatocellular carcinoma (HCC), by using PlGF knockout mice and monoclonal anti-PlGF antibodies in a mouse model for HCC. In addition, the effect of PlGF antibodies is compared to that of sorafenib, as well as the combination of both therapies.

Results: We have found that both in a transgenic knockout model and in a treatment model, targeting PlGF significantly decreases tumor burden. This was achieved not only by inhibiting neovascularisation, but also by decreasing hepatic macrophage recruitment and by normalising the remaining blood vessels, thereby decreasing hypoxia and reducing the prometastatic potential of HCC.

Conclusions: Considering the favourable safety profile and its pleiotropic effect on vascularisation, metastasis and inflammation, PlGF inhibition could become a valuable therapeutic strategy against HCC.
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http://dx.doi.org/10.1016/j.jhep.2012.09.032DOI Listing
February 2013

Serum protein N-glycan alterations of diethylnitrosamine-induced hepatocellular carcinoma mice and their evolution after inhibition of the placental growth factor.

Mol Cell Biochem 2013 Jan 24;372(1-2):199-210. Epub 2012 Sep 24.

Department of Hepatology and Gastroenterology, Ghent University Hospital, 9000 Ghent, Belgium.

Placental growth factor (PlGF) inhibition produced promising results in reducing tumor burden in a diethylnitrosamine (DEN)-induced mouse model for hepatocellular carcinoma (HCC). The aim of this study was to non-invasively assess the improved histology by performing a serum glycomic analysis. To elucidate the molecular mechanism underlying the observed glycomic effects, we investigated the transcription and expression of E26 transformation-specific sequence 1 (Ets-1), a transcription factor essential for the glycomic and angiogenic changes in malignant transformation, including its different phosphorylated forms that result from activation of the MAP kinase and a Ca(2+)-dependent pathway. In addition, three Ets-1-dependent glycosyltransferase genes, Mgat4a, Mgat4b, and Mgat5, were also evaluated. HCC was induced in mice by weekly injections with DEN for 16, 20, 25, and 30 w. In the treatment study, mice were injected with DEN for 25 w and subsequently treated with PlGF antibodies (5D11D4) for 5 w. Finally, PlGF-/- mice were injected with DEN for 20, 25, and 30 w. Serum N-glycans were analyzed with DNA sequencer-assisted fluorophore-assisted capillary electrophoresis and compared with histology. Maximum altered N-glycan phenotype was reached after 20 w of DEN-injections, i.e., when the first neoplastic lesions started to appear. 5D11D4-treatment improved the glycomic phenotype in that 7 of the 11 altered glycans tended to normalize. The PlGF-/- mice also showed a normalization trend, although not to the same extent of the treatment group. Number of Ets1, Mgat4a, Mgat4b, and Mgat5 transcripts increased considerably in DEN-injected mice, however, a non-significant decrease was observed after 5D11D4-treatment. On the protein level, 5D11D4-treatment had a prominent effect on the MAP kinase pathway with a significant p38 activation, yet independent of Ets-1 function.
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http://dx.doi.org/10.1007/s11010-012-1461-1DOI Listing
January 2013

Inhibition of the placental growth factor decreases burden of cholangiocarcinoma and hepatocellular carcinoma in a transgenic mouse model.

Eur J Gastroenterol Hepatol 2012 Sep;24(9):1020-32

Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium.

Objectives: Hepatocellular carcinoma and cholangiocarcinoma form the majority of primary hepatic tumours and are the third most common cause of cancer-related deaths. These liver tumours rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors and triggering the angiogenic switch. Therefore, inhibiting angiogenesis has proven to be a valuable therapeutic strategy in hepatocellular carcinoma, yet less is known about its use in cholangiocarcinoma. In this study, we assess whether inhibiting the placental growth factor (PlGF) could offer a therapeutic option in mice with hepatocellular carcinoma and cholangiocarcinoma. PlGF is a homologue of the vascular endothelial growth factor, which is only involved in pathological angiogenesis, therefore, its inhibition does not induce adverse effects.

Methods: We have used a chemically induced transgenic mouse model in which both hepatocellular carcinoma and cholangiocarcinoma develop after 25 weeks and are treated with murine monoclonal antibodies targeting PlGF.

Results: This study has shown for the first time that inhibiting PlGF decreases the burden of cholangiocarcinoma, by affecting both angiogenesis and inflammation.

Conclusion: The use of monoclonal antibodies targeting PlGF could thus offer a potential systemic treatment for patients who suffer from primary liver tumours.
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http://dx.doi.org/10.1097/MEG.0b013e3283554219DOI Listing
September 2012

Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease.

Cytokine 2012 Aug 31;59(2):442-9. Epub 2012 May 31.

Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium.

The liver is a major target of injury in obese patients. Non-alcoholic fatty liver disease (NAFLD) is present in 60-90% of obese Americans and can range from simple steatosis to the more severe non-alcoholic steatohepatitis (NASH). The onset of a chronic inflammatory reaction marks the progression from simple steatosis to NASH and the expansion of adipose tissue is strongly associated with angiogenesis. Therefore, we determined the serum concentration of inflammatory [tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6)] and angiogenic [vascular endothelial growth factor A (VEGF)] cytokines and soluble VEGF receptors 1 and 2 (sVEGFR1, sVEGFR2) in the serum of an obese population with simple steatosis and NASH compared to healthy controls. Moreover, we determined the TNFα, IL6, VEGF, VEGFR1 and VEGFR2 gene expression in the liver of these simple steatosis and NASH patients. The population consisted of 30 obese patients, which were diagnosed with simple steatosis and 32 patients with NASH and compared to 30 age-and-sex matched healthy controls. Mean serum TNFα levels were elevated in the serum of simple steatosis and NASH patients compared to healthy controls, reaching significance in NASH patients. IL6 was significantly increased in simple steatosis and NASH patients compared to the healthy controls. VEGF levels were significantly elevated in patients with simple steatosis and borderline significantly elevated in NASH patients compared to the serum levels of healthy control subjects. The concentration of sVEGFR1 was significantly increased in serum of simple steatosis and NASH patients compared to controls. sVEGFR2 concentration was not significantly different in the three groups. TNFα mRNA expression was higher in NASH patients compared to simple steatosis patients. Hepatic gene expression of VEGF, VEGFR1 and VEGFR2 were slightly decreased in NASH patients compared to simple steatosis patients. These data indicate the involvement of inflammatory (TNFα and IL6), angiogenic (VEGF) cytokines and sVEGFR1 in the pathophysiology of NAFLD.
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http://dx.doi.org/10.1016/j.cyto.2012.05.001DOI Listing
August 2012

Effect of prolyl hydroxylase domain-2 haplodeficiency on the hepatocarcinogenesis in mice.

J Hepatol 2012 Jul 13;57(1):61-8. Epub 2012 Mar 13.

Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium.

Background & Aims: The two major primary liver cancers in adults are hepatocellular carcinoma and cholangiocarcinoma. These tumors rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors. Recently, interest has grown in the regulators of these factors. Several reports have been published describing the role of prolyl hydroxylase domains--the key oxygen sensor responsible for the degradation of hypoxia inducible factors--tumor progression and vascularisation. The effect of prolyl hydroxylase domain 2 on the pathogenesis of liver cancer has never been studied.

Methods: A diethylnitrosamine-induced mouse model was used in this study, allowing primary hepatic tumors to occur as a result of chronic liver damage. Several parameters of prolyl hydroxylase domain 2-haplodeficient mice were compared to those of wild type mice, thereby focussing on the expression of angiogenic factors and on the hepatic progenitor cell activation and differentiation.

Results: This study shows that inhibiting prolyl hydroxylase domain 2 increases the hepatocarcinogenesis and stimulates the development of cholangiocarcinoma. Furthermore, PHD2 deficiency and the accompanying continuous HIF activation, selected for a more metastatic tumor phenotype.

Conclusions: The effect of prolyl hydroxylase domain 2 deficiency on hepatocarcinogenesis hold a great potential for therapeutic intervention, since hypoxia and the selection for a more aggressive cholangiocarcinoma phenotype might also have a repercussion on patients receiving long-term treatment with anti-angiogenic compounds.
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http://dx.doi.org/10.1016/j.jhep.2012.02.021DOI Listing
July 2012

Inhibition of placental growth factor activity reduces the severity of fibrosis, inflammation, and portal hypertension in cirrhotic mice.

Hepatology 2011 May;53(5):1629-40

Department of Hepatology and Gastroenterology, Ghent University Hospital, Ghent, Belgium.

Unlabelled: Placental growth factor (PlGF) is associated selectively with pathological angiogenesis, and PlGF blockade does not affect the healthy vasculature. Anti-PlGF is therefore currently being clinically evaluated for the treatment of cancer patients. In cirrhosis, hepatic fibrogenesis is accompanied by extensive angiogenesis. In this paper, we evaluated the pathophysiological role of PlGF and the therapeutic potential of anti-PlGF in liver cirrhosis. PlGF was significantly up-regulated in the CCl(4) -induced rodent model of liver cirrhosis as well as in cirrhotic patients. Compared with wild-type animals, cirrhotic PlGF(-/-) mice showed a significant reduction in angiogenesis, arteriogenesis, inflammation, fibrosis, and portal hypertension. Importantly, pharmacological inhibition with anti-PlGF antibodies yielded similar results as genetic loss of PlGF. Notably, PlGF treatment of activated hepatic stellate cells induced sustained extracellular signal-regulated kinase 1/2 phosphorylation, as well as chemotaxis and proliferation, indicating a previously unrecognized profibrogenic role of PlGF.

Conclusion: PlGF is a disease-candidate gene in liver cirrhosis, and inhibition of PlGF offers a therapeutic alternative with an attractive safety profile.
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http://dx.doi.org/10.1002/hep.24238DOI Listing
May 2011

Angiogenesis in chronic liver disease and its complications.

Liver Int 2011 Feb 15;31(2):146-62. Epub 2010 Nov 15.

Department of Hepatology and Gastroenterology, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.

Nowadays, liver cancer, cirrhosis and other liver-related diseases are the fifth most common cause of mortality in the UK. Furthermore, chronic liver diseases (CLDs) are one of the major causes of death, which are still increasing year-on-year. Therefore, knowledge about the pathophysiology of CLDs and its complications is of uttermost importance. The goal of this review is to clarify the role of angiogenesis in the disease progression of various liver diseases. Looking closer at the pathophysiology of portal hypertension (PH), fibrosis, cirrhosis, non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC), we find that angiogenesis is a recurring factor in the disease progression. In PH, several factors involved in its pathogenesis, such as hypoxia, oxidative stress, inflammation and shear stress are potential mediators for the angiogenic response. The progression from fibrosis to cirrhosis, the end-point of CLDs, is distinguished by a prolonged inflammatory and fibrogenic process that leads to an abnormal angioarchitecture distinctive for cirrhosis. In several stages of NASH, a link might be made between the disease progression and hepatic microvasculature changes. HCC is one of the most vascular solid tumours in which angiogenesis plays an important role in its development, progression and metastasis. The close relationship between the progression of CLDs and angiogenesis emphasises the need for anti-angiogenic therapy as a tool for blocking or slowing down the disease progression. The fact that angiogenesis plays a pivotal role in CLDs gives rise to new opportunities for treating CLDs and its complications.
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http://dx.doi.org/10.1111/j.1478-3231.2010.02369.xDOI Listing
February 2011

Kinetics of angiogenic changes in a new mouse model for hepatocellular carcinoma.

Mol Cancer 2010 Aug 20;9:219. Epub 2010 Aug 20.

Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium.

Background: The increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest of scientific research in this field. Therefore, a vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma (HCC) in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for HCC research, in which tumours develop in a natural background of fibrosis and (2) to assess the time-dependent angiogenic changes in the pathogenesis of HCC.

Methods: Weekly intraperitoneal injections with the hepatocarcinogenic compound N-nitrosodiethylamine was applied as induction method and samples were taken at several time points to assess the angiogenic changes during the progression of HCC.

Results: The N-nitrosodiethylamine-induced mouse model provides well vascularised orthotopic tumours after 25 weeks. It is a representative model for human HCC and can serve as an excellent platform for the development of new therapeutic targets.
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http://dx.doi.org/10.1186/1476-4598-9-219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2936339PMC
August 2010

Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Cell 2010 Apr;141(1):178-90

Vesalius Research Center, KU Leuven, Leuven B-3000, Belgium.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.
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http://dx.doi.org/10.1016/j.cell.2010.02.039DOI Listing
April 2010

Experimental mouse models for hepatocellular carcinoma research.

Int J Exp Pathol 2009 Aug;90(4):367-86

Department of Gastroenterology and Hepatology, Ghent University Hospital, 9000 Ghent, Belgium.

Every year almost 500,000 new patients are diagnosed with hepatocellular carcinoma (HCC), a primary malignancy of the liver that is associated with a poor prognosis. Numerous experimental models have been developed to define the pathogenesis of HCC and to test novel drug candidates. This review analyses several mouse models useful for HCC research and points out their advantages and weaknesses. Chemically induced HCC mice models mimic the injury-fibrosis-malignancy cycle by administration of a genotoxic compound alone or, if necessary, followed by a promoting agent. Xenograft models develop HCC by implanting hepatoma cell lines in mice, either ectopically or orthotopically; these models are suitable for drug screening, although extrapolation should be considered with caution as multiple cell lines must always be used. The hollow fibre assay offers a solution for limiting the number of test animals in xenograft research because of the ability for implanting multiple cell lines in one mouse. There is also a broad range of genetically modified mice engineered to investigate the pathophysiology of HCC. Transgenic mice expressing viral genes, oncogenes and/or growth factors allow the identification of pathways involved in hepatocarcinogenesis.
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http://dx.doi.org/10.1111/j.1365-2613.2009.00656.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2741148PMC
August 2009