Comparative Analysis of Proteins in Extracellular Vesicles from Quiescent versus Activated Hepatic Stellate Cells

Abstract

AimsA newly recognized mechanism of cell communication is intercellular trafficking of molecules that are contained within nano‐sized extracellular vesicles (EVs) such as exosomes or microvesicles. EVs mediate cell‐cell transfer of their cargo (miRs, mRNAs, proteins) resulting in functional reprograming of recipient cells. Hepatic stellate cells (HSC) are a normally‐quiescent cell type in the liver but after hepatic injury they transdifferentiate into collagen‐producing myofibroblasts that account for the majority of the fibrotic scar that is deposited during chronic liver injury. Recent studies have shown that EVs from quiescent HSC have anti‐fibrogenic properties whereas those from activated HSC are pro‐fibrogenic. To help understand these distinct properties, proteins in EVs from quiescent versus activated HSC were evaluated by mass spectrometry.MethodsNanoparticle tracking and Western blot confirmed the presence of EVs after differential ultracentrifugation of serum‐free conditioned medium from quiescent (Day 1–4) or activated (passage 1 (P1)) primary mouse HS. Five separate D4 EV preparations and three separate P1 EV preparations were individually subjected to mass spectrometry. Data were analyzed using Mascot (Matrix Science, London, UK) while MS/MS based peptide and protein identifications were validated with Scaffold (Proteome Software Inc., Portland, OR). Peptide identification was accepted if there was > 95% probability of FDR < 1.0% by the Peptide Prophet algorithm with Scaffold delta‐mass correction. Protein identifications were accepted if there was >99.0% probability of FDR < 1.0% with at least 2 constituent peptides identified. Protein probabilities were assigned by the Protein Prophet algorithm.Results46 proteins were identified in at least three of five D4 EV samples while 337 proteins were identified in three P1 EV samples. D4 EVs contained 19 specific proteins while P1 EVs contained 310 specific proteins. There were 27 proteins that were shared between both groups, with 11 of these proteins present at significantly higher levels in D4 EVs compared to P1 EVs. EVs from both groups contained exosomal proteins, but D4 EVs exhibited higher abundance of nucleosome components (histones, 17.4%, 46.7‐fold enrichment) or keratin filaments (15.2%, 57.6‐fold enrichment) while P1 EVs contained more proteins associated with extracellular space (30.9%, 4.0‐fold), proteasome complex (6.8%, 24.3‐fold), basement membrane (6.5%, 14.5‐fold), and collagen trimer (3.9%, 10.0‐fold). KEGG analysis showed 1 unique pathway for D4 EVs, 33 unique pathways for P1 EVs and 4 shared pathways for D4 and P1 EVs.ConclusionsEVs produced by activated HSC contain a protein cargo that is qualitatively and quantitatively different from that of EVs from quiescent HSC. These differences may account for distinct biological responses when each EV population acts on various hepatic cells, thus contributing to their differential pathophysiological or therapeutic outcomes. RC and XL contributed equally to this work.Support or Funding InformationNIH AA023626 & AA025974This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.