Proteomic changes in extracellular vesicles from hepatocytes after cytotoxic ethanol injury

Aims Extracellular vesicles (EVs) play an important role in maintaining normal homeostasis or driving pathogenesis in the liver. For example, various disease‐associated processes (e.g. steatosis, inflammation, fibrosis) are, on the one hand, promoted by EVs produced by hepatocytes after cell stress, injury, or infection yet, on the other hand, inhibited by EVs produced by normal hepatocytes. To better understand these functional differences, proteomic analysis was performed on EVs produced by AML12 mouse hepatocytes maintained in vitro under normal conditions or after exposure to ethanol and TNFα. Methods AML12 cells were cultured under serum‐free conditions either alone or in the presence of 50mM ethanol for 48 hours plus 30ng/ml TNF‐α over the latter 24 hrs. EVs were purified from conditioned medium by sequential steps of low‐, high‐ and ultra‐ centrifugation. Proteins from two separate EV preparations collected under control (EV Norm ) or ethanol/TNFα (EV ETOH/TNF ) culture conditions were extracted, digested with trypsin, and protein identification was performed using nano‐liquid chromatography‐nanospray tandem mass spectrometry (LC/MS/MS). Sequence data were searched against the Uniprot Mouse database using Mascot Daemon by Matrix Science version 2.6.0 (Boston, MA) Proteins with less than 1% FDR as well as a minimal of 2 significant peptides detected were considered as valid proteins. Results 293 proteins were present in duplicate EV Norm samples and 186 proteins were present in duplicate EV ETOH/TNF samples. 152 proteins were shared by both EV Norm and EV ETOH/TNF and were present at relatively higher levels (quantitative value 10–1000) than the 141 EV Norm ‐specific proteins (quantitative value 5–50) or the 34 EV ETOH/TNF ‐specific proteins (quantitative values 1–10). Among the most abundant shared proteins, FN, CLUS, ACTG, KPYM and TERA showed diminished levels in EV ETOH/TNF versus EV Norm while LG3BP, CFAB, H2AY, RS27A, and IGSF8 showed increased levels in EV ETOH/TNF versus EV Norm . GO analysis showed that of the proteins that were unique or enriched in EV Norm or EV ETOH/TNF , some components were shared (exosome, membrane, cytoplasm, nucleus, focal adhesion, extracellular components, cell‐cell adherins, nucleolus, ribonucleoproteins) while other components were unique to either EV Norm (cytoskeleton, mitochondria, myelin sheath, protein complex) or EV ETOH/TNF (ribosome, endocytosis, cytosol, Golgi). KEGG analysis showed that the principal pathways for EV Norm ‐specific/enriched proteins related to antibiotic synthesis, carbon metabolism, proteasome, actin cytoskeleton, virus infection, focal adhesion, PI3K‐AKT, amino acid metabolism, and glycolysis whereas EV ETOH/TNF ‐specific/enriched proteins related to ribosome, endocytosis, proteasome, system lupus erythematosus, alcoholism and mineral absorption. Conclusions EVs from normal or ethanol/TNFα ‐treated hepatocytes show dynamic differences in their respective proteomes. The underlying changes in EV protein composition, both quantitatively and qualitatively, likely contribute to the role that these EV populations play in regulating hepatic homeostasis and the pathogenic outcomes following cytotoxic ethanolic injury in the liver. Support or Funding Information NIH grants R21 AA025974 and R21 AA023626