Modeling system states in liver cells: Survival, apoptosis and their modifications in response to viral infection

<p>Abstract</p> <p>Background</p> <p>The decision pro- or contra apoptosis is complex, involves a number of different inputs, and is central for the homeostasis of an individual cell as well as for the maintenance and regeneration of the complete organism.</p> <p>Results</p> <p>This study centers on Fas ligand (FasL)-mediated apoptosis, and a complex and internally strongly linked network is assembled around the central FasL-mediated apoptosis cascade. Different bioinformatical techniques are employed and different crosstalk possibilities including the integrin pathway are considered. This network is translated into a Boolean network (74 nodes, 108 edges). System stability is dynamically sampled and investigated using the software SQUAD. Testing a number of alternative crosstalk possibilities and networks we find that there are four stable system states, two states comprising cell survival and two states describing apoptosis by the intrinsic and the extrinsic pathways, respectively. The model is validated by comparing it to experimental data from kinetics of cytochrome c release and caspase activation in wildtype and Bid knockout cells grown on different substrates. Pathophysiological modifications such as input from cytomegalovirus proteins M36 and M45 again produces output behavior that well agrees with experimental data.</p> <p>Conclusion</p> <p>A network model for apoptosis and crosstalk in hepatocytes shows four different system states and reproduces a number of different conditions around apoptosis including effects of different growth substrates and viral infections. It produces semi-quantitative predictions on the activity of individual nodes, agreeing with experimental data. The model (SBML format) and all data are available for further predictions and development.</p

Modeling of signaling pathways in different biological systems

Die Apoptose der Leberzellen ist abhängig von externen Signalen wie beispielsweise Komponenten der Extrazellulären Matrix sowie anderen Zell-Zell-Kontakten, welche von einer Vielfalt und Vielzahl an Knoten verarbeitet werden. Einige von ihnen wurden im Rahmen dieser Arbeit auf ihre Systemeffekte hin unter- sucht. Trotz verschiedener äußerer Einflüsse und natürlicher Selektion ist das System daraufhin optimiert, eine kleine Anzahl verschiedener und klar voneinander unterscheidbarer Systemzustände anzunehmen. Die verschiedenartigen Einflüsse und Crosstalk-Mechanismen dienen der Optimierung der vorhandenen Systemzustände. Das in dieser Arbeit vorgestellte Modell zeigt zwei apoptotische sowie zwei nicht-apoptotische stabile Systemzustände, wobei der Grad der Aktivierung eines Knotens bis zu dem Moment stark variieren kann, in welchem der absolute Systemzustand selbst verändert wird (Philippi et al., BMC Systems Biology,2009) [1]. Dieses Modell stellt zwar eine Vereinfachung des gesamten zellulären Netzwerkes und seiner verschiedenen Zustände dar, ist aber trotz allem in der Lage, unabhängig von detaillierten kinetischen Daten und Parametern der einzelnen Knoten zu agieren. Gleichwohl erlaubt das Modell mit guter qualitativer Übereinstimmung die Apoptose als Folge einer Stimulation mit FasL zu modellieren. Weiterhin umfasst das Modell sowohl Crosstalk-Möglichkeiten des Collagen-Integrin-Signalwegs, ebenso berücksichtigt es die Auswirkungen der genetischen Deletion von Bid sowie die Konsequenzen einer viralen Infektion. In einem zweiten Teil werden andere Anwendungsmöglichkeiten dargestellt. Hormonale Signale in Pflanzen, Virusinfektionen und intrazelluläre Kommunikation werden semi-quantitativ modelliert. Auch hier zeigte sich eine gute Ubereinstimmung der Modelle mit den experimentellen Daten.Apoptosis of liver cells is dependent on external signals such as components of the extracellular matrix and cell-cell-contacts, which are processed by a variety of numerous nodes of which several are examined here for their system effects. Despite different input interferences and presumably also due to natural selecti- on, the system nevertheless appears to be optimized to adopt a small number of clear and distinguishable states, and the various inputs and crosstalk mechanisms only optimize the best choice between them. For the model described within this work, two nonapoptotic and two apoptotic states are found, although the degree of activation at a node can differ widely until the absolute system state is altered (Philippi et al., BMC Systems Biology, 2009) [1]. The model is still a simplification of the complete cellular network and its different states, and operates independently of detailed kinetic data and parameters for individual nodes. Nevertheless, it allows modeling the readout of apoptosis after FasL stimulation with qualitative agreement and includes crosstalks from collagen/integrin signa- ling, the effect of genetic deletion of Bid and the consequences of viral infection. The second part of this work deals with other applications using this method. Semi-quantitative models are used for hormonal signaling in plants, viral infec- tions and intra-cellular communication. The simulated results fit to the experi- mental data provided

Histological comparison of arterial thrombi in mice and men and the influence of Cl-amidine on thrombus formation

<div><p>Aims</p><p>Medical treatment of arterial thrombosis is mainly directed against platelets and coagulation factors, and can lead to bleeding complications. Novel antithrombotic therapies targeting immune cells and neutrophil extracellular traps (NETs) are currently being investigated in animals. We addressed whether immune cell composition of arterial thrombi induced in mouse models of thrombosis resemble those of human patients with acute myocardial infarction (AMI).</p><p>Methods and results</p><p>In a prospective cohort study of patients suffering from AMI, 81 human arterial thrombi were harvested during percutaneous coronary intervention and subjected to detailed histological analysis. In mice, arterial thrombi were induced using two distinct experimental models, ferric chloride (FeCl₃) and wire injury of the carotid artery. We found that murine arterial thrombi induced by FeCl₃ were highly concordant with human coronary thrombi regarding their immune cell composition, with neutrophils being the most abundant cell type, as well as the presence of NETs and coagulation factors. Pharmacological treatment of mice with the protein arginine deiminase (PAD)-inhibitor Cl-amidine abrogated NET formation, reduced arterial thrombosis and limited injury in a model of myocardial infarction.</p><p>Conclusions</p><p>Neutrophils are a hallmark of arterial thrombi in patients suffering from acute myocardial infarction and in mouse models of arterial thrombosis. Inhibition of PAD could represent an interesting strategy for the treatment of arterial thrombosis to reduce neutrophil-associated tissue damage and improve functional outcome.</p></div

NETs in arterial thrombi of mice and humans.

<p>(A) Representative illustration of NETs stained for NE and DNA (DAPI) in the early phase of arterial thrombosis. Human and mouse thrombi showed comparable morphology after 3, 6 or 12h. Extracellular DNA originates from NE+ neutrophils. Bars, 10μm. Arrows, nuclei; arrowheads, NET fibers. (B) Quantification of NETs per 100 neutrophils in human thrombi (<12h) (n = 10) and experimental thrombosis (FeCl₃) (3–6h) (n = 5). Dots represent individual experiments; lines indicate mean values for each group. (C) Association between thrombus age and number of NETs in mice and humans.</p

Characteristics of human arterial thrombi.

<p>(A) Pie chart shows the distribution of thrombus age. 50 out of 81 patients described the precise onset of AMI symptoms, which allowed the calculation of thrombus age following its removal during PCI. The majority of human thrombi (with precise onset of symptoms) was younger than 24h. (B) Leukocyte accumulation in human thrombi. Representative images of HE staining (n = 3). Bars, 200μm (top image) and 50μm (bottom image). (C) Immunohistochemical visualization of leukocytes (CD45, green, n = 3), neutrophils (NE, red, n = 81) and monocytes (CD14, green, n = 11). Nuclei are counterstained with Hoechst (including controls). Control (isotype) or secondary antibody alone. Bars, 10μm. (D) The graph shows the quantification of monocytes (n = 11) and neutrophils (n = 81) in human thrombi. Results are shown as mean ± SD. (E) Correlation between human thrombi younger than 12h and the number of leukocytes (n = 33).</p

List of antibodies used for immunohistochemistry.

<p>List of antibodies used for immunohistochemistry.</p

Characteristics of mouse arterial thrombi induced by FeCl₃ injury or wire denudation in mice.

<p>(A) Immunohistological images of platelet aggregate area (red) in arterial thrombi (n = 3/group) and control stainings. Bars, 100μm. Control (isotype) or secondary antibody alone. (B) Comparison of leukocyte recruitment to the mouse carotid artery 3h after FeCl₃ exposure or wire denudation (n = 3/group). Representative images show immunohistochemical staining for leukocytes (CD45, green) and their subsets, as distinguished by expression of neutrophil elastase (NE, red) for neutrophils and CD68 (red) for blood monocytes. Nuclei were counterstained with Hoechst (including controls). Bars, 10μm. Control (isotype) or secondary antibody alone. (C) Association between number of leukocytes and thrombus age (n = 3/group). Mean ± SD. (D) Quantification of monocyte and neutrophil subsets within mouse thrombi 3h after FeCl₃ exposure (n = 3/group). Mean ± SD.</p