Extracellular vesicle-mediated transfer of functional RNA in the tumor microenvironment.

Extracellular vesicles (EVs) have been shown to transfer various molecules, including functional RNA between cells and this process has been suggested to be particularly relevant in tumor-host interactions. However, data on EV-mediated RNA transfer has been obtained primarily by in vitro experiments or involving ex vivo manipulations likely affecting its biology, leaving their physiological relevance unclear. We engineered glioma and carcinoma tumor cells to express Cre recombinase showing their release of EVs containing Cre mRNA in various EV subfractions including exosomes. Transplantation of these genetically modified tumor cells into mice with a Cre reporter background leads to frequent recombination events at the tumor site. In both tumor models the majority of recombined cells are CD45+ leukocytes, predominantly Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs). In addition, multiple lineages of recombined cells can be observed in the glioma model. In the lung carcinoma model, recombined MDSCs display an enhanced immunosuppressive phenotype and an altered miRNA profile compared to their non-recombined counterparts. Cre-lox based tracing of tumor EV RNA transfer in vivo can therefore be used to identify individual target cells in the tumor microenvironment for further mechanistical or functional analysis.

Comparative analysis and modeling of the severity of steatohepatitis in DDC-treated mouse strains.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has a broad spectrum of disease states ranging from mild steatosis characterized by an abnormal retention of lipids within liver cells to steatohepatitis (NASH) showing fat accumulation, inflammation, ballooning and degradation of hepatocytes, and fibrosis. Ultimately, steatohepatitis can result in liver cirrhosis and hepatocellular carcinoma. METHODOLOGY AND RESULTS: In this study we have analyzed three different mouse strains, A/J, C57BL/6J, and PWD/PhJ, that show different degrees of steatohepatitis when administered a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) containing diet. RNA-Seq gene expression analysis, protein analysis and metabolic profiling were applied to identify differentially expressed genes/proteins and perturbed metabolite levels of mouse liver samples upon DDC-treatment. Pathway analysis revealed alteration of arachidonic acid (AA) and S-adenosylmethionine (SAMe) metabolism upon other pathways. To understand metabolic changes of arachidonic acid metabolism in the light of disease expression profiles a kinetic model of this pathway was developed and optimized according to metabolite levels. Subsequently, the model was used to study in silico effects of potential drug targets for steatohepatitis. CONCLUSIONS: We identified AA/eicosanoid metabolism as highly perturbed in DDC-induced mice using a combination of an experimental and in silico approach. Our analysis of the AA/eicosanoid metabolic pathway suggests that 5-hydroxyeicosatetraenoic acid (5-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE) and prostaglandin D2 (PGD2) are perturbed in DDC mice. We further demonstrate that a dynamic model can be used for qualitative prediction of metabolic changes based on transcriptomics data in a disease-related context. Furthermore, SAMe metabolism was identified as being perturbed due to DDC treatment. Several genes as well as some metabolites of this module show differences between A/J and C57BL/6J on the one hand and PWD/PhJ on the other.

Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation.

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

Gene expression profiling unravels cancer-related hepatic molecular signatures in steatohepatitis but not in steatosis.

BACKGROUND: Pathogenesis and factors for determining progression of alcoholic and non-alcoholic steatosis to steatohepatitis with risk of further progression to liver cirrhosis and cancer are poorly understood. In the present study, we aimed to identify potential molecular signatures for discrimination of steatohepatitis from steatosis. METHODOLOGY AND RESULTS: Global microarray gene expression analysis was applied to unravel differentially expressed genes between steatohepatitis compared to steatosis and control samples. For functional annotation as well as the identification of disease-relevant biological processes of the differentially expressed genes the gene ontology (GO) database was used. Selected candidate genes (n = 46) were validated in 87 human liver samples from two sample cohorts by quantitative real-time PCR (qRT-PCR). The GO analysis revealed that genes down-regulated in steatohepatitis were mainly involved in metabolic processes. Genes up-regulated in steatohepatitis samples were associated with cancer progression and proliferation. In surgical liver resection samples, 39 genes and in percutaneous liver biopsies, 30 genes were significantly up-regulated in steatohepatitis. Furthermore, immunohistochemical investigation of human liver tissue revealed a significant increase of AKR1B10 protein expression in steatohepatitis. CONCLUSIONS: The development of steatohepatitis is characterized by distinct molecular changes. The most striking examples in this respect were KRT23 and AKR1B10, which we found to be highly differentially expressed in steatohepatitis compared to steatosis and normal liver. We propose that KRT23 and AKR1B10 may serve as future potential biomarkers for steatohepatitis as well as markers for progression to HCC.

Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.

Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.