Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.
OBJECTIVE
Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC).


METHODS
MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats.


RESULTS
Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL.


CONCLUSIONS
Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.
Witek
Rafal P
RP
Department of Medicine, Duke University, Durham, North Carolina 27710, USA.
Yang
Liu
L
Liu
Renshui
R
Jung
Youngmi
Y
Omenetti
Alessia
A
Syn
Wing-Kin
WK
Choi
Steve S
SS
Cheong
Yeiwon
Y
Fearing
Caitlin M
CM
Agboola
Kolade M
KM
Chen
Wei
W
Diehl
Anna Mae
AM
D000818:Animals; D000077214:Becaplermin; D001652:Bile Ducts / Q000502:physiology; D055252:Cell-Derived Microparticles / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000502:physiology*; D015870:Gene Expression; D053823:Hedgehog Proteins / Q000502:physiology*; D055166:Hepatic Stellate Cells / Q000502:physiology; D022781:Hepatocytes / Q000502:physiology*; D006975:Hypertension, Portal / Q000209:etiology; D008026:Ligation; D008103:Liver Cirrhosis / Q000473:pathology*; D008297:Male; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*
D053823:Hedgehog Proteins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin