Pathways of production and delivery of hepatocyte exosomes

Hepatocyte exosomes (Exo^Hep) are proposed to mediate physiological or pathophysiological signaling in a variety of hepatic target cells. Exo^Hep were purified from the medium of primary mouse hepatocytes or AML12 cells and characterized as ~100 nm nanovesicles that were positive for proteins commonly found in exosomes (CD9, CD81, flotillin) or hepatocytes (asialoglycoprotein receptor). Ethanol treatment of hepatocytes caused increased Exo^Hep release and increased cellular mRNA expression of components involved in intracellular vesicle trafficking (Rab 5a,b,c, Rab 7a, Rab 27a,b) or exosome biogenesis via the ESCRT (HGS, Alix, STAM1, TSG101, VTA1, YKT6) or ceramide (nSmase2) pathways. RNA interference of HGS, Alix, TSG101 or nSmase 2 caused exosome production by normal or ethanol-treated hepatocytes to be reduced. In mice, in vivo administration of fluorescently-labeled Exo^Hep resulted in their accumulation in the liver and preferential localization to hepatic stellate cells (HSC) or hepatocytes, the latter of which showed enhanced Exo^Hep binding when isolated from fibrotic mice. In cell co-cultures, the intercellular transfer of RNA from hepatocytes to hepatocytes or HSC was blocked by the exosome inhibitor GW4869. Exo^Hep binding to HSC or hepatocytes occurred via mechanisms that involved heparin-like molecules and cellular integrin αv or β1 subunits , and resulted in a reversal of fibrosis-associated gene expression in HSC and of ethanol-induced damage in hepatocytes. These studies provide insight regarding the regulation and/or participation of exosome biogenesis or trafficking components in hepatocytes and show that Exo^Hep can mediate therapeutic changes in activated HSC or injured hepatocytes that occur downstream of heparin- or integrin-dependent binding interactions.