L. Chen1, R. Chen1, D. Brigstock1,2 1Nationwide Children’s Hospital,Pediatric Surgery,Columbus, OH, USA 2Ohio State University,Pediatric Surgery,Columbus, OH, USA
Introduction: A key event in chronic liver injury is the activation of hepatic stellate cells (HSC) which causes these normally quiescent cells to become proliferative, to express alpha-smooth muscle actin (αSMA), and to perpetually synthesize fibrogenic mediators such as connective tissue growth factor (CTGF), resulting in unrelentingly deposition of collagen which causes fibrosis and ultimately compromises liver function. The goal of this work was to establish if pathways of HSC fibrogenesis are regulated by hepatocyte-derived exosomes. Exosomes (which arise by inward budding of the limiting membrane of multivesicular bodies (MVB)) are membranous nanovesicles which function as intercellular molecular shuttles for delivering their complex payload (microRNA, mRNA protein) to other cells.
Methods: Exosomes that had been isolated from cultures of primary mouse hepatocytes or AML12 mouse hepatocytes were stained with PKH26 fluorescent dye and incubated with primary mouse HSC for up to 48 hrs after which HSC-associated fluorescence was evaluated. GFP-transfected hepatocytes, treated with or without the exosome inhibitor GW4869, were co-cultured with HSC in which GFP uptake was assessed. mRNA encoding exosome biogenic components was evaluated by RT-PCR in hepatocytes after exposure of the cells to 0-50mM ethanol. Expression of αSMA or CTGF in HSC was evaluated by RT-PCR following their 48-hr treatment with exosomes from control AML12 hepatocytes, or from AML12 cells that had been treated with 50mM ethanol for 48hr, or 30ng/ml TNFα for the last 24hrs or ethanol for 48hrs and TNFα for the last 24hrs.
Results: Expression of nSMase2 (required for biosynthesis of ceramide on which exosome production depends), Rab5b/c (regulates biogenesis of precursors of MVB), and/or Rab27a/c (promotes fusion of exosome-laden MVBs with the plasma membrane) was dose-dependently stimulated in hepatocytes by ethanol. PKH26-stained hepatocyte-derived exosomes were taken up by HSC. GW4869-mediated inhibition of nSMase2 in GFP-transfected AML12 cells caused reduced delivery of exosomal GFP to recipient HSC without affecting donor cell GFP expression. Fibrogenic activity or activation in HSC was not stimulated by exosomes from healthy hepatocytes whereas exosomes from ethanol-sensitized hepatocytes exposed to TNFα stimulated αSMA or CTGF expression, an effect that was more pronounced as compared to the response to exosomes from hepatocytes exposed to each stimulant alone.
Conclusion: Exosomes from hepatocytes are able to bind to HSC with the result that, if the exosomes are from injured hepatocytes, they can stimulate HSC activation and fibrogenesis. These results identify exosomes as molecular shuttles which can transmit pro-fibrogenic molecular cues from injured hepatocytes to HSC. This identifies a novel mechanism of hepatocyte-HSC cross-talk and provides a new lead for developing novel strategies for liver fibrosis therapy.