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Pathways of production and delivery of hepatocyte exosomes

  • Research Article
  • Published:
Journal of Cell Communication and Signaling Aims and scope

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.

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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

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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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Authors and Affiliations

  1. Center for Clinical and Translational Research, The Research Institute at Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, OH, 43205, USA

    Li Chen, Ruju Chen, Sherri Kemper & David R. Brigstock

  2. Department of Surgery, The Ohio State University, Columbus, OH, 43210, USA

    David R. Brigstock

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  1. Li Chen
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  2. Ruju Chen
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Correspondence to David R. Brigstock.

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Chen, L., Chen, R., Kemper, S. et al. Pathways of production and delivery of hepatocyte exosomes. J. Cell Commun. Signal. 12, 343–357 (2018). https://doi.org/10.1007/s12079-017-0421-7

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  • DOI: https://doi.org/10.1007/s12079-017-0421-7

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