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Journal of Cell Communication and Signaling

, Volume 12, Issue 1, pp 343–357 | Cite as

Pathways of production and delivery of hepatocyte exosomes

  • Li Chen
  • Ruju Chen
  • Sherri Kemper
  • David R. BrigstockEmail author
Research Article

Abstract

Hepatocyte exosomes (ExoHep) are proposed to mediate physiological or pathophysiological signaling in a variety of hepatic target cells. ExoHep 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 ExoHep 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 ExoHep resulted in their accumulation in the liver and preferential localization to hepatic stellate cells (HSC) or hepatocytes, the latter of which showed enhanced ExoHep 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. ExoHep 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 ExoHep can mediate therapeutic changes in activated HSC or injured hepatocytes that occur downstream of heparin- or integrin-dependent binding interactions.

Keywords

Alcohol Ceramide ESCRT Extracellular vesicle Hepatic stellate cell Liver 

Abbreviations

αSMA

Alpha smooth muscle actin

ASGPR1

Asialoglycoprotein receptor 1

CCl4

Carbon tetrachloride

CCN2

Connective tissue growth factor

DAPI

4′,6-Diamidine-2′-phenylindole dihydrochloride

EDTA

Ethylenediaminetetraacetic acid

ESCRT

Endosomal sorting complex required for transport

ExoHep

Exosomes from normal hepatocytes

ExoHep-TNFα/EtOH

Exosomes from TNFα-primed ethanol-treated hepatocytes

EV

Extracellular vesicle

FBS

Fetal bovine serum

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HSC

Hepatic stellate cell

KC

Kupffer cell

LSEC

Luminal sinusoidal endothelial cells

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MVB

Multivesicular bodies

nSmase 2

Neutral sphingomyelinase 2

NTA

Nanoparticle tracking analysis

PBS

Phosphate-buffered saline

qRT-PCR

Quantitative real time polymerase chain reaction

siRNA

Small interfering RNA

TEM

Transmission electron microscopy

TGF-β

Transforming growth factor beta

TNFα

Tumor necrosis factor alpha

Notes

Acknowledgements

This work was supported by NIH grants R01AA021276, R21AA023626, and R21AA025974 awarded to D.R.B. and by pilot funding to D.R.B from NIH grant P50AA024333 in support of the Northern Ohio Alcohol Center (Principal Investigator, Laura Nagy, PhD). We thank David Dunaway and Victoria Velazquez for assistance with cell sorting and NTA, and Cindy McAllister for help with TEM.

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Copyright information

© The International CCN Society 2017

Authors and Affiliations

  1. 1.Center for Clinical and Translational ResearchThe Research Institute at Nationwide Children’s HospitalColumbusUSA
  2. 2.Department of SurgeryThe Ohio State UniversityColumbusUSA

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