Sebastian Zellmer

Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration

Only little is known about how cells coordinately behave to establish functional tissue structure and restore microarchitecture during regeneration. Research in this field is hampered by a lack of techniques that allow quantification of tissue architecture and its development. To bridge this gap, we have established a procedure based on confocal laser scans, image processing, and three-dimensional tissue reconstruction, as well as quantitative mathematical modeling. As a proof of principle, we reconstructed and modeled liver regeneration in mice after damage by CCl4, a prototypical inducer of pericentral liver damage. We have chosen the regenerating liver as an example because of the tight link between liver architecture and function: the complex microarchitecture formed by hepatocytes and microvessels, i.e. sinusoids, ensures optimal exchange of metabolites between blood and hepatocytes. Our model captures all hepatocytes and sinusoids of a liver lobule during a 16 days regeneration process. The model unambiguously predicted a so-far unrecognized mechanism as essential for liver regeneration, whereby daughter hepatocytes align along the orientation of the closest sinusoid, a process which we named “hepatocyte-sinusoid alignment” (HSA). The simulated tissue architecture was only in agreement with the experimentally obtained data when HSA was included into the model and, moreover, no other likely mechanism could replace it. In order to experimentally validate the model of prediction of HSA, we analyzed the three-dimensional orientation of daughter hepatocytes in relation to the sinusoids. The results of this analysis clearly confirmed the model prediction. We believe our procedure is widely applicable in the systems biology of tissues.

Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy

The PI(3)K–PKB–FOXO signalling network provides a major intracellular hub for the regulation of cell proliferation, survival and stress resistance. Here we report an unexpected role for FOXO transcription factors in regulating autophagy by modulating intracellular glutamine levels. To identify transcriptional targets of this network, we performed global transcriptional analyses after conditional activation of the key components PI(3)K, PKB/Akt, FOXO3 and FOXO4. Using this pathway approach, we identified glutamine synthetase as being transcriptionally regulated by PI(3)K–PKB–FOXO signalling. Conditional activation of FOXO also led to an increased level of glutamine production. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes in a glutamine-synthetase-dependent manner. This resulted in an increased level of autophagy as measured by LC3 lipidation, p62 degradation and fluorescent imaging of multiple autophagosomal markers. Inhibition of FOXO3-mediated autophagy increased the level of apoptosis, suggesting that the induction of autophagy by FOXO3-mediated glutamine synthetase expression is important for cellular survival. These findings reveal a growth-factor-responsive network that can directly modulate autophagy through the regulation of glutamine metabolism.

Interaction of phosphatidylcholine liposomes with the human stratum corneum

The interaction of dimyristoylphosphatidylcholine liposomes with the human stratum corneum was investigated by confocal laser scanning microscopy and differential scanning calorimetry. Human skin is characterized by a high autofluorescence. By introducing appropriate optical filters the autofluorescence of the skin was depressed and the penetration profile of fluorescence labelled vesicles was investigated. From optical sectioning it was obvious that neither the vesicles nor the fluorophore N-(lissamine rhodamine B sulfonyl)diacylphophatidylethanolamine (Rho-PE) penetrates in detectable amounts into the human skin. Differential scanning calorimetry of human stratum corneum revealed, that the peak positions of the human stratum corneum specific endothermic transitions at 10 degrees C, 35 degrees C, 50 degrees C, 62 degrees C, 73 degrees C and 81 degrees C did not change significantly after 18 h of non-occlusive vesicle application. However, the enthalpy of the transitions at 35 degrees C, 50 degrees C, 62 degrees C and 73 degrees C, estimated through peak heights increased, relative to the protein related peak at 81 degrees C. A novel transition at 10 degrees C was observed. From these data we conclude that DMPC liposomes do not penetrate intact into the human skin. We deduce, however, that the vesicles disintegrate at the surface of stratum corneum after non-occlusive application. The individual lipid molecules then interact with the lipid barrier of the stratum corneum and penetrate into the latter, which results in an increase of the enthalpy, related to the lipid components of the SC.

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)

Transcription factors ETF, E2F, and SP-1 are involved in cytokine-independent proliferation of murine hepatocytes.

The cellular basis of liver regeneration has been intensely investigated for many years. However, the mechanisms initiating hepatocyte “plasticity” and priming for proliferation are not yet fully clear. We investigated alterations in gene expression patterns during the first 72 hours of C57BL/6N mouse hepatocyte culture on collagen monolayers (CM), which display a high basal frequency of proliferation in the absence of cytokines. Although many metabolic genes were down‐regulated, genes related to mitogen‐activated protein kinase (MAPK) signaling and cell cycle were up‐regulated. The latter genes showed an overrepresentation of transcription factor binding sites (TFBS) for ETF (TEA domain family member 2), E2F1 (E2F transcription factor 1), and SP‐1 (Sp1 transcription factor) (P < 0.001), all depending on MAPK signaling. Time‐dependent increase of ERK1/2 phosphorylation occurred during the first 48 hours (and beyond) in the absence of cytokines, accompanied by an enhanced bromodeoxyuridine labeling index of 20%. The MEK inhibitor PD98059 blunted these effects indicating MAPK signaling as major trigger for this cytokine‐independent proliferative response. In line with these in vitro findings, liver tissue of mice challenged with CCl4 displayed hepatocytes with intense p‐ERK1/2 staining and nuclear SP‐1 and E2F1 expression. Furthermore, differentially expressed genes in mice after partial hepatectomy contained overrepresented TFBS for ETF, E2F1, and SP‐1 and displayed increased expression of E2F1. Conclusion: Cultivation of murine hepatocytes on CM primes cells for proliferation through cytokine‐independent activation of MAPK signaling. The transcription factors ETF, E2F1, and SP‐1 seem to play a pronounced role in mediating proliferation‐dependent differential gene expression. Similar events, but on a shorter time‐scale, occur very early after liver damage in vivo. (HEPATOLOGY 2010;.)

Profiling of human stratum corneum ceramides by liquid chromatography-electrospray mass spectrometry

Abstract The investigation of the role of ceramides in maintaining the barrier function of stratum corneum (SC) has brought about the need of specific analytical methods. The present paper describes a combination of high-performance thin-layer chromatography (HPTLC) using automated multiple development, which was modified for semi-preparative purposes, with subsequent liquid chromatography–electrospray mass spectrometry for the profiling of ceramides. The fractionation of complex lipid extracts with the help of thin-layer chromatography facilitates a specific, sensitive and reliable analysis. Characteristic patterns of the molecular mass distribution in different ceramide fractions are easily obtainable. Additional electrospray tandem mass spectrometry using an ion trap mass spectrometer provides fragment ions which indicate the sphingoid base as well as the fatty acid moiety and therefore enables the exact assignment of ceramide structures. Furthermore, a method of liquid chromatography–electrospray mass spectrometry for the quantitation of cholesterol-3-sulfate is presented.

A Systematic Evaluation of the Use of Physiologically Based Pharmacokinetic Modeling for Cross-Species Extrapolation

Transfer of knowledge along the different phases of drug development is a fundamental process in pharmaceutical research. In particular, cross-species extrapolation between different laboratory animals and further on to first-in-human trials is challenging because of the uncertain comparability of physiological processes. Physiologically based pharmacokinetic (PBPK) modeling allows translation of mechanistic knowledge from one species to another by specifically considering physiological and biochemical differences in between. We here evaluated different knowledge-driven approaches for cross-species extrapolation by systematically incorporating specific model parameter domains of a target species into the PBPK model of a reference species. Altogether, 15 knowledge-driven approaches were applied to murine and human PBPK models of 10 exemplary drugs resulting in 300 different extrapolations. Statistical analysis of the quality of the different extrapolations revealed not only species-specific physiology as the key determinant in cross-species extrapolation but also identified a synergistic effect when considering both kinetic rate constants and gene expression profiles of relevant enzymes and transporters. Moreover, we show that considering species-specific physiology, plasma protein binding, enzyme and transport kinetics, as well as tissue-specific gene expression profiles in PBPK modeling increases accuracy of cross-species extrapolations and thus supports first-in-human trials based on prior preclinical knowledge.

Individual variation of human plantar stratum corneum lipids, determined by automated multiple development of high-performance thin-layer chromatography plates

The stratum corneum lipids are unique in composition and have been used frequently as a model system of the skins lipid barrier. Automated multiple development (AMD) of high-performance thin-layer chromatography plates in combination with a 25-step gradient, based on methanol, diethyl ether and n-hexane separated the six major human plantar stratum corneum lipids. Post-chromatographic staining of these lipids with a solution of MnCl2-H2SO4 at 130 degrees C or a solution of CuSO4-H3PO4 at 140 degrees C allowed visualization of the lipids and quantification. The MnCl2-H2SO4 solution stained saturated fatty acids less intensely. Therefore, the CuSO4-H3PO4 solution was used for quantification and we found, on average, 2.06% (w/w) cholesterol 3-sulphate, 20.16% (w/w) free fatty acids, 20.25% (w/w) ceramides, 43.53% (w/w) non-esterified sterols, 4.56% (w/w) triacylglycerols and 9.4% (w/w) sterolesters in the human plantar stratum corneum extracts. The concentration of phospholipids was less than 1% (w/w). In addition, the lipid composition of twenty different human plantar stratum corneum extracts was determined. Statistics revealed a correlation between the ratio of free fatty acids and non-esterified sterols (r=0.832, p<0.01, n=20). Several control experiments proved that this correlation is not due to the extraction method, the post-chromatographic staining procedure or bacterial contamination of the stratum corneum.

Model-guided identification of a therapeutic strategy to reduce hyperammonemia in liver diseases

BACKGROUND & AIMS Recently, spatial-temporal/metabolic mathematical models have been established that allow the simulation of metabolic processes in tissues. We applied these models to decipher ammonia detoxification mechanisms in the liver. METHODS An integrated metabolic-spatial-temporal model was used to generate hypotheses of ammonia metabolism. Predicted mechanisms were validated using time-resolved analyses of nitrogen metabolism, activity analyses, immunostaining and gene expression after induction of liver damage in mice. Moreover, blood from the portal vein, liver vein and mixed venous blood was analyzed in a time dependent manner. RESULTS Modeling revealed an underestimation of ammonia consumption after liver damage when only the currently established mechanisms of ammonia detoxification were simulated. By iterative cycles of modeling and experiments, the reductive amidation of alpha-ketoglutarate (α-KG) via glutamate dehydrogenase (GDH) was identified as the lacking component. GDH is released from damaged hepatocytes into the blood where it consumes ammonia to generate glutamate, thereby providing systemic protection against hyperammonemia. This mechanism was exploited therapeutically in a mouse model of hyperammonemia by injecting GDH together with optimized doses of cofactors. Intravenous injection of GDH (720 U/kg), α-KG (280 mg/kg) and NADPH (180 mg/kg) reduced the elevated blood ammonia concentrations (>200 μM) to levels close to normal within only 15 min. CONCLUSION If successfully translated to patients the GDH-based therapy might provide a less aggressive therapeutic alternative for patients with severe hyperammonemia.

Oxidative damage of human skin lipids: Dependence of lipid peroxidation on sterol concentration

Photoprotection against sunburn and associated irradiation-induced damages of the human skin is mainly attributed to the darkening of the biochrome melanin by its oxidation. Human skin lipids were examined for an additional protection by sterols. Lipid vesicles prepared from extracted human skin lipids as well as from mixtures of typical lipids of the stratum corneum were irradiated by UV light in the presence and absence of oxygen. The oxidative degradation of various lipids was measured by quantitative HPTLC, by the dichlorofluorescein fluorescent assay, by the thiobarbituric acid assay and a novel luminol-based chemiluminescence technique. Electron spin resonance was used to look for certain radical intermediates. The results indicate, that sterols, mainly free cholesterol, with their high concentration in the lipid barrier of the stratum corneum (up to 50 mol%) effectively compete with the peroxidation of other human skin lipids (ceramides and free fatty acids).