Elena Blázquez-López, Félix Royo, Elena Vázquez-Ogando, Francisco Javier Cubero, Raquel Herrero, Laura Moreno, José A. Lorente, Angel Cogolludo, Rafael Correa, Marjorie Pion, Yulia Nevzorova, Johanna Sierra, Marta Puerto, Ismael Ranz, Agustin Albillos, Jaime Bosch, Rafael Bañares, Jordi Gracia-Sancho, Juan Falcon-Perez, Javier Vaquero.
ABSTRACT: THU 168
Background and Aims: Most information about hepatocytederived exosomes and extracellular vesicles (EVs) and their role in health and disease derives from cell culture experiments, which do not represent the complexity of whole organisms. The lineage reporter mT/mG mouse strain allows the generation of mice that express the Tomato-dye protein in the plasma membrane of all cells except in Cre-recombinase expressing cells, which express the Green Fluorescent Protein (EGFP). Our Aim was to evaluate the lineage reporter mT/mG mouse strain in experimental liver disease as a novel approach for the in vivo identification of circulating EVs according to the cell of origin.
Method: The mT/mG mouse strain (Stock 07676, Jackson Lab) was crossbred with mice expressing Cre-recombinase under the control of the Lysozyme-C (LysMCrexmT/mG mice, EGFP in myeloid-derived cells) or the Albumin promoters (AlbCrexmT/mG mice, EGFP in hepatocytes). Non-fluorescent mice (C57Bl6/J) and the original mT/mGmouse strainwere used as controls. Hepatic damagewas induced by acetaminophen administration (APAP, 300 mg/kg bwip). EVswere isolated by immune-magnetic separation (Mouse Exosome Isolation Kit Pan [CD9, CD63 and CD81 proteins] or GFP Isolation Kit, Miltenyi) from mouse plasma, and by ultracentrifugation of culture media from primary hepatocyte cultures from each mouse strain. EVs were analyzed by flow cytometry (FACS, MACSQuant16), Nanoparticle Tracking Analysis (NTA), Dynamic Light Scattering (DLS), transmission electron microscopy (TEM) and Western Blotting.
Results: The plasma concentration of EGFP-positive EVs was low in Vehicle-treated AlbCrexmT/mG mice but it largely increased in APAPtreated mice, as assessed by FACS. In contrast, APAP treatment did not change the plasma concentration of EGFP-positive EVs in
LysMCrexmT/mG mice. EGFP-positivity of EVs from AlbCrexmT/mG micewas confirmed by FACS in plasma and hepatocyte culture media samples, and by Western blotting in exosomes isolated from both sources. In mouse plasma samples, the immune-magnetic isolation
of hepatocyte-derived and myeloid cell-derived EGFP-positive EVs was also confirmed by FACS andWestern blotting. The characteristics of exosome population were confirmed in all EVs isolates.
Conclusion: The circulating concentration of hepatocyte-derived EVs is low in normal animals, but it considerably increases after acute hepatic insults. The lineage reporter mT/mG mouse strain allows the evaluation and isolation of hepatocyte-derived and myeloid cellderived
EVs in murine models of liver disease. This highly valuable new approach for the in vivo study of the cell-type of origin of EVs overcomes amajor limitation in EVs research, being also applicable to fields other than Hepatology.
ARTICLE: Research Gate