Hepcidin knockout mice fed with iron-rich diet develop chronic liver injury and liver fibrosis due to lysosomal iron overload.

BACKGROUND & AIMS: Hepcidin is the central regulator of iron homeostasis and altered hepcidin signalling results in both hereditary and acquired iron overload. While the association between iron overload and development of end-stage liver disease is well established, the underlying mechanisms are largely unknown. To improve that, we analysed hepcidin knockout (KO) mice as a model of iron overload-associated liver disease. METHODS: Hepcidin wild type (WT) and KO mice fed with 3% carbonyl iron-containing diet starting at one month of age were compared to age-matched animals kept on standard chow. Liver histology and serum parameters were used to assess the extent of liver injury and fibrosis. Iron distribution was determined by subcellular fractionation and electron microscopy. RESULTS: Among mice kept on iron-rich diet, 6 months old hepcidin KO mice (vs. WT) displayed profound hepatic iron overload (3,186 ± 411 vs. 1,045 ± 159 µg/mg tissue, p<0.005), elevated liver enzymes (ALT: KO 128 ± 6, WT 56 ± 5 IU/L, p<0.05), mild hepatic inflammation and hepatocellular apoptosis. Twelve, but not six months old KO mice fed with iron-rich diet developed moderate liver fibrosis. The liver injury was accompanied by a marked lysosomal iron overload and lysosomal fragility with release of cathepsin B into the cytoplasm. Increased p62 levels and autofluorescent iron complexes suggested impaired protein degradation. As a mechanism leading to lysosomal iron overload, the autophagy (lysosomal influx) was increased. CONCLUSIONS: Hepcidin KO mice represent a novel model of iron overload-related liver diseases and implicate lysosomal injury as a crucial event in iron toxicity.

CHOP-mediated hepcidin suppression modulates hepatic iron load.

The liver is the central regulator of iron metabolism and accordingly, chronic liver diseases often lead to systemic iron overload due to diminished expression of the iron-regulatory hormone hepcidin. To study the largely unknown regulation of iron metabolism in the context of hepatic disease, we used two established models of chronic liver injury, ie repeated carbon tetrachloride (CCl(4)) or thioacetamide (TAA) injections. To determine the impact of CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP) on hepcidin production, the effect of a single TAA injection was determined in wild-type and CHOP knockout mice. Furthermore, CHOP and hepcidin expression was assessed in control subjects and patients with alcoholic liver disease. Both chronic injury models developed a distinct iron overload in macrophages. TAA-, but not CCl(4) - injected mice displayed additional iron accumulation in hepatocytes, resulting in a significant hepatic and systemic iron overload which was due to suppressed hepcidin levels. C/EBPα signalling, a known hepcidin inducer, was markedly inhibited in TAA mice, due to lower C/EBPα levels and overexpression of CHOP, a C/EBPα inhibitor. A single TAA injection resulted in a long-lasting (> 6 days) suppression of hepcidin levels and CHOP knockouts (compared to wild-types) displayed significantly attenuated hepcidin down-regulation in response to acute TAA administration. CHOP mRNA levels increased 5-fold in alcoholic liver disease patients versus controls (p < 0.005) and negatively correlated with hepcidin expression. Our results establish CHOP as an important regulator of hepatic hepcidin expression in chronic liver disease. The differences in iron metabolism between the two widely used fibrosis models likely reflect the differential regulation of hepcidin expression in human liver disease.

Image-derived mean velocity measurement for prediction of coronary flow reserve in a canonical stenosis phantom using magnetic particle imaging.

INTRODUCTION: Aim of this study is to evaluate whether magnetic particle imaging (MPI) is capable of measuring velocities occurring in the coronary arteries and to compute coronary flow reserve (CFR) in a canonical phantom as a preliminary study. METHODS: For basic velocity measurements, a circulation phantom was designed containing replaceable glass tubes with three varying inner diameters, matching coronary-vessel diameters. Standardised boluses of superparamagnetic-iron-oxide-nanoparticles were injected and visualised by MPI. Two image-based techniques were competitively applied to calibrate the respective glass tube and to compute the mean velocity: full-duration-at-half-maximum (FDHM) and tracer dilution (TD) method. For CFR-calculation, four necessary settings of the circulation model of a virtual vessel with an inner diameter of 4 mm were generated using differently sized glass tubes and a stenosis model. The respective velocities in stenotic glass tubes were computed without recalibration. RESULTS: On velocity level, comparison showed a good agreement (rFDHM = 0.869, rTD = 0.796) between techniques, preferably better for 4 mm and 6 mm inner diameter glass tubes. On CFR level MPI-derived CFR-prediction performed considerably inferior with a relative error of 20-44%. CONCLUSIONS: MPI has the ability to reliably measure coronary blood velocities at rest as well as under hyperaemia and therefore may be suitable for CFR calculation. Calibration-associated accuracy of CFR-measurements has to be improved substantially in further studies.

Discovery of a Highly Selective Tankyrase Inhibitor Displaying Growth Inhibition Effects against a Diverse Range of Tumor Derived Cell Lines.

The availability of high quality probes for specific protein targets is fundamental to the investigation of their function and their validation as therapeutic targets. We report the utilization of a dedicated chemoproteomic assay platform combining affinity enrichment technology with high-resolution protein mass spectrometry to the discovery of a novel nicotinamide isoster, the tetrazoloquinoxaline 41, a highly potent and selective tankyrase inhibitor. We also describe the use of 41 to investigate the biology of tankyrase, revealing the compound induced growth inhibition of a number of tumor derived cell lines, demonstrating the potential of tankyrase inhibitors in oncology.

A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level.

Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement in vivo. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels-Alder reaction between trans-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors.

Clinical and cellular features in patients with primary autosomal recessive microcephaly and a novel CDK5RAP2 mutation.

BACKGROUND: Primary autosomal recessive microcephaly (MCPH) is a rare neurodevelopmental disorder that results in severe microcephaly at birth with pronounced reduction in brain volume, particularly of the neocortex, simplified cortical gyration and intellectual disability. Homozygous mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2 are the cause of MCPH3. Despite considerable interest in MCPH as a model disorder for brain development, the underlying pathomechanism has not been definitively established and only four pedigrees with three CDK5RAP2 mutations have been reported. Specifically for MCPH3, no detailed radiological or histological descriptions exist. METHODS/RESULTS: We sought to characterize the clinical and radiological features and pathological cellular processes that contribute to the human MCPH3 phenotype. Haplotype analysis using microsatellite markers around the MCPH1-7 and PNKP loci in an Italian family with two sons with primary microcephaly, revealed possible linkage to the MCPH3 locus. Sequencing of the coding exons and exon/intron splice junctions of the CDK5RAP2 gene identified homozygosity for the novel nonsense mutation, c.4441C > T (p.Arg1481*), in both affected sons. cMRI showed microcephaly, simplified gyral pattern and hypogenesis of the corpus callosum. The cellular phenotype was assessed in EBV-transformed lymphocyte cell lines established from the two affected sons and compared with healthy male controls. CDK5RAP2 protein levels were below detection level in immortalized lymphocytes from the patients. Moreover, mitotic spindle defects and disrupted γ-tubulin localization to the centrosome were apparent. CONCLUSION: These results suggest that spindle defects and a disruption of centrosome integrity play an important role in the development of microcephaly in MCPH3.

The iron-regulatory peptide hepcidin is upregulated in the ischemic and in the remote myocardium after myocardial infarction.

Recent evidence suggests that iron metabolism contributes to the ischemic damage after myocardial infarction. Hepcidin, a recently discovered peptide hormone, regulates iron uptake and metabolism, protecting the body from iron overload. In this study we analyzed the regulation of hepcidin in the heart and blood of rats after myocardial infarction. To induce a myocardial infarction in the rats, left anterior descending coronary artery ligation was performed. After 1-24h, biopsies from the ischemic and the non-ischemic myocardium were taken. In these biopsies, the mRNA levels and the protein expression of hepcidin were analyzed by quantitative RT-PCR and immunoblot analysis, respectively. In parallel, the serum levels of prohepcidin were measured by ELISA. Six hours after myocardial infarction, the hepcidin mRNA expression was temporally upregulated in the ischemic and in the non-ischemic myocardium. The upregulation was specific for hepcidin, since other iron-related genes (hemojuvelin, IREG-1) remained unchanged. Furthermore, the alteration of the hepcidin protein expression in the ischemic area was connected to the level of hepcidin in the serum of the infarcted rats, where hepcidin also raised up. Angiotensin receptor blockade with candesartan did not influence the mRNA regulation of hepcidin. Together, these data show a particular upregulation of the iron-regulatory peptide hepcidin in the ischemic and the non-ischemic myocardium after myocardial infarction. It is speculated that upregulation of hepcidin may reduce iron toxicity and thus infarct size expansion in an infarcted heart.

Influence of irradiation on metabolism and metastatic potential of B16-F10 melanoma cells.

PURPOSE: To analyse short term and long term X-ray irradiation effects on proliferation, viability, glucose and amino acid uptake of murine melanoma cells in vitro and metastasis in vivo. MATERIALS AND METHODS: B16-F10 melanoma cells were irradiated with different doses of X-ray irradiation (200 kV) in the range from 1-20 Gy. One, two and three days respectively 7, 14 and 21 days after treatment cells were analysed concerning cell growth, viability, proliferation, cell cycle distribution, glucose and amino acid transport. Moreover the capability of the cells for in vivo metastasis was examined. RESULTS: As short term response on irradiation we detected decreased cell growth, viability and arrest in the G2/M phase of the cell cycle. Long term response involves re-start of proliferation, increased cell growth and glucose uptake but still decreased viability and amino acid transport. In vivo metastasis is lost immediately after irradiation and regained to a low extent beyond two weeks time for recurrence of cells before injection. CONCLUSIONS: In vitro data suggest that surviving melanoma cells compensate the initial irradiation-dependent damage of proliferation within three weeks possibly by increase in glucose uptake. For metastasis in vivo the role of additional mechanisms is strongly suggested.