J Böttger

Integrated metabolic spatial-temporal model for the prediction of ammonia detoxification during liver damage and regeneration

The impairment of hepatic metabolism due to liver injury has high systemic relevance. However, it is difficult to calculate the impairment of metabolic capacity from a specific pattern of liver damage with conventional techniques. We established an integrated metabolic spatial‐temporal model (IM) using hepatic ammonia detoxification as a paradigm. First, a metabolic model (MM) based on mass balancing and mouse liver perfusion data was established to describe ammonia detoxification and its zonation. Next, the MM was combined with a spatial‐temporal model simulating liver tissue damage and regeneration after CCl4 intoxication. The resulting IM simulated and visualized whether, where, and to what extent liver damage compromised ammonia detoxification. It allowed us to enter the extent and spatial patterns of liver damage and then calculate the outflow concentrations of ammonia, glutamine, and urea in the hepatic vein. The model was validated through comparisons with (1) published data for isolated, perfused livers with and without CCl4 intoxication and (2) a set of in vivo experiments. Using the experimentally determined portal concentrations of ammonia, the model adequately predicted metabolite concentrations over time in the hepatic vein during toxin‐induced liver damage and regeneration in rodents. Further simulations, especially in combination with a simplified model of blood circulation with three ammonia‐detoxifying compartments, indicated a yet unidentified process of ammonia consumption during liver regeneration and revealed unexpected concomitant changes in amino acid metabolism in the liver and at extrahepatic sites. Conclusion: The IM of hepatic ammonia detoxification considerably improves our understanding of the metabolic impact of liver disease and highlights the importance of integrated modeling approaches on the way toward virtual organisms. (Hepatology 2014;;60:2039–2050)

“Artificial micro organs”—a microfluidic device for dielectrophoretic assembly of liver sinusoids

In order to study possible toxic side effects of potential drug compounds in vitro a reliable test system is needed. Predicting liver toxicity presents a major challenge of particular importance as liver cells grown in a cell culture suffer from a rapid loss of their liver specific functions. Therefore we are developing a new microfluidic test system for liver toxicity. This test system is based on an organ-like liver 3D co-culture of hepatocytes and endothelial cells. We devised a microfluidic chip featuring cell culture chambers with integrated electrodes for the assembly of liver sinusoids by dielectrophoresis. Fluid channels enable an organ-like perfusion with culture media and test compounds. Different chamber designs were studied and optimized with regard to dielectrophoretic force distribution, hydrodynamic flow profile, and cell trapping rate using numeric simulations. Based on simulation results a microchip was injection-moulded from COP. This chip allowed the assembly of viable hepatocytes and endothelial cells in a sinusoid-like fashion.

Hepatic Hedgehog signaling contributes to the regulation of IGF1 and IGFBP1 serum levels

BackgroundHedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial.FindingsTwo types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels.ConclusionsOur results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

Response of sinusoidal mouse liver cells to choline-deficient ethionine-supplemented diet

BackgroundProliferation of oval cells, the bipotent precursor cells of the liver, requires impeded proliferation and loss of hepatocytes as well as a specific micro-environment, provided by adjacent sinusoidal cells of liver. Despite their immense importance for triggering the oval cell response, cells of hepatic sinusoids are rarely investigated. To elucidate the response of sinusoidal liver cells we have employed a choline-deficient, ethionine-supplemented (CDE) diet, a common method for inducing an oval cell response in rodent liver. We have utilised selected expression markers commonly used in the past for phenotypic discrimination of oval cells and sinusoidal cells: cytokeratin, E-cadherin and M2-pyruvate kinase for oval cells; and glial fibrillary acidic protein (GFAP) was used for hepatic stellate cells (HSCs).ResultsCDE diet leads to an activation of all cells of the hepatic sinusoid in the mouse liver. Beside oval cells, also HSCs and Kupffer cells proliferate. The entire fraction of proliferating cells in mouse liver as well as endothelial cells and cholangiocytes express M2-pyruvate kinase. Concomitantly, GFAP, long considered a unique marker of quiescent HSCs was upregulated in activated HSCs and expressed also in cholangiocytes and oval cells.ConclusionsOur results point to an important role of all types of sinusoidal cells in regeneration from CDE induced liver damage and call for utmost caution in using traditional marker for identifying specific cell types. Thus, M2-pyruvate kinase should no longer be used for estimating the oval cell response in mouse liver. CDE diet leads to activation of GFAP positive HSCs in the pericentral zone of liver lobulus. In the periportal zone the detection of GFAP in biliary cells and oval cells, calls other cell types as progenitors of hepatocytes into question under CDE diet conditions.

Dual origin, development, and fate of bovine pancreatic islets

Endocrine cells are evident at an early stage in bovine pancreatic development when the pancreas still consists of primitive epithelial cords. At this stage, the endocrine cells are interspersed between the precursor cells destined to form the ductulo‐acinar trees of later exocrine lobules. We here demonstrate that, in bovine fetuses of crown rump length ≥ 11 cm, the endocrine cells become increasingly segregated from the developing exocrine pancreas by assembly into two units that differ in histogenesis, architecture, and fate. Small numbers of ‘perilobular giant islets’ are distinguishable from larger numbers of ‘intralobular small islets’. The two types of islets arise in parallel from the ends of the ductal tree. Aside from differences in number, location, and size, the giant and small islets differ in cellular composition (predominantly insulin‐synthesising cells vs. mixtures of endocrine cells), morphology (epithelial trabeculae with gyriform and rosette‐like appearance vs. compact circular arrangements of endocrine cells), and in their relationships to intrapancreatic ganglia and nerves. A further difference becomes apparent during the antenatal period; while the ‘interlobular small islets’ persist in the pancreata of calves and adult cattle, the perilobular giant islets are subject to regression, characterised by involution of the parenchyma, extensive haemorrhage, leukocyte infiltration (myeloid and T‐cells) and progressive fibrotic replacement. In conclusion, epithelial precursor cells of the ductolo‐acinar tree may give rise to populations of pancreatic islets with different histomorphology, cellular composition and fates. This should be taken into account when using these cells for the generation of pancreatic islets for transplantation therapy.

Tailoring microfluidic systems for organ-like cell culture applications using multiphysics simulations

Replacing animal testing with in vitro cocultures of human cells is a long-term goal in pre-clinical drug tests used to gain reliable insight into drug-induced cell toxicity. However, current state-of-the-art 2D or 3D cell cultures aiming at mimicking human organs in vitro still lack organ-like morphology and perfusion and thus organ-like functions. To this end, microfluidic systems enable construction of cell culture devices which can be designed to more closely resemble the smallest functional unit of organs. Multiphysics simulations represent a powerful tool to study the various relevant physical phenomena and their impact on functionality inside microfluidic structures. This is particularly useful as it allows for assessment of system functions already during the design stage prior to actual chip fabrication. In the HepaChip®, dielectrophoretic forces are used to assemble human hepatocytes and human endothelial cells in liver sinusoid-like structures. Numerical simulations of flow distribution, shear stress, electrical fields and heat dissipation inside the cell assembly chambers as well as surface wetting and surface tension effects during filling of the microchannel network supported the design of this human-liver-on-chip microfluidic system for cell culture applications. Based on the device design resulting thereof, a prototype chip was injection-moulded in COP (cyclic olefin polymer). Functional hepatocyte and endothelial cell cocultures were established inside the HepaChip® showing excellent metabolic and secretory performance.

Towards Artificial Liver Sinusoids by Dielectrophoretic Cell Assembly in Microfluidic System for Use in Substance Screening

We are developing microfluidic systems with 3D-microstructures and integrated electrodes to assemble cells in an organ-like 3D-structure by means of dielectrophoretic and hydrodynamic forces. To further mimic the in vivo situation cells are allowed to adhere on liver specific extra cellular matrix proteins. Results showing co-assembly of liver cells in a sinusoid like structure will be presented.

Das Netzwerk zwischen dem Wnt/β-Catenin und dem Hedgehog Signalweg

Hintergrund: Es ist bekannt, dass es haufig zu Crosstalk-Reaktionen und Feedbackloops zwischen diversen Komponenten verschiedener Signalkaskaden kommt. Diese Situation trifft auch auf die evolutionar konservierten, morphogenen Signalwege Wnt/β-Catenin und Hedgehog zu, welche sich in den unterschiedlichsten Geweben und Zellen gegenseitig beeinflussen konnen. Fur uns ist es interessant zu erfahren, ob diese Wechselwikungen auch fur primare Hepatozyten zutreffen. Methoden: Um mogliche Beziehungen zwischen den beiden Signalwegen offenlegen zu konnen, wurde von uns die Aktivitat des einen Signalwegs mittels siRNA-vermitteltem Knockdown variiert und der Einfluss auf die transkriptionelle Aktivitat der unterschiedlichsten Komponenten des jeweils anderen Signalwegs bestimmt. So wurden, nachdem wir mittels RNAi die Expression von Hedgehog Genen, wie z.B. SuFu, Ptch und Smo in primaren Maushepatocyten ausgeschalten haben, die die mRNA-Spiegel von Komponenten des Wnt/β-Catenin Signalwegs mit q-RT-PCR analysiert. Umgekehrt wurde durch den siRNA Knockdown des APC der Wnt/β-Catenin Signalweg aktiviert und anschliesend die mRNA-Spiegel von Komponenten des Hedgehog Signalwegs experimentell bestimmt Ergebnisse: Die transkriptionelle Antwort des Wnt/β-Catenin Signalweges auf die Veranderungen in der Hedgehog Kaskade waren sehr vielgestaltig. Je nach Art der Beeinflussung des Signalweges, d.h. ob der Hedgehog in seiner Aktivitat nach oben bzw. nach unten reguliert wurde, reagierten auch die Gene im Wnt/β-Catenin Signalweg unterschiedlich. So wurde die Expression von Axin2, APC und weiteren Wnt/β-Catenin Genen durch das Ausschalten von SuFu und der daraus resultierenden Intensivierung des Hedgehog Signalweges, signifikant verringert. Eine ahnliche Abnahme der mRNA-Spiegel verzeichneten wir beim Knockdown von Smo und den Transkripionsfaktoren Gli1, 2 und 3, wohingegen bei den RNAi Versuchen mit Ptch1 und Ptch2 eine Zunahme in den Expressionen zu erkennen waren. Bei der Beeinflussung der Wnt/β-Catenin Kaskade durch die Verringerung der APC mRNA, konnten wir eine Verminderung in der Expression von Gli1, 2 und 3 und eine Zunahme von Ihh detektieren. Zusammenfassung: Anhand dieser Ergebnisse lasst sich zusammenfassend, dass es in adulten Maushepatozyten einen intensiven Crosstalk zwischen den beiden Signalkaskaden Wnt/β-Catenin und Hedgehog auf transkriptioneller Ebene zu geben scheint.