Liver Cell–Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells

Abstract

Background & Aims 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-HSCs and cholangiocytes were exposed to platelet-derived growth factor (PDGF) to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy (TEM) and immunoblots, and applied to Hh-reporter containing cells. Microparticles were also 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, a Hh signaling inhibitor. Effects on SEC gene expression were evaluated by QRT-PCR and immunoblotting. Finally, Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Results PDGF-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically active Hh ligands. BDL also increased release of Hh-containing exosome-enriched microparticles into plasma and bile. TEM 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.

Hepatic sinusoids in liver injury, inflammation, and fibrosis: new pathophysiological insights

“…PDGF-treated HSC and cholangiocytes release exosomes with hedgehog ligands, which induce hedgehog-dependent changes in SEC with upregulation of several genes leading to an angiogenic phenotype. Of note, bile duct ligation in mice increased the release of such exosomes, too, supporting further evidence for a key role of extracellular vesicles in liver fibrogenesis [76]. While HSC communicate with SEC via exosomes, the same seems to be true vice versa.…”

“…Hedgehog ligands are known to activate downstream pathways in endothelial cells through exosomes during embryogenesis [75]. In a recent study, Witek et al [76] showed that those hedgehog ligands play an important role in HSC-SEC crosstalk and angiogenesis via exosomeenriched microparticles. PDGF-treated HSC and cholangiocytes release exosomes with hedgehog ligands, which induce hedgehog-dependent changes in SEC with upregulation of several genes leading to an angiogenic phenotype.…”

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration

“…In vitro studies indicate that exosomes may up-regulate the expression of fibrolytic matrix metalloproteinase genes in HSCs (42,43). In other studies, exosomes were capable of transducing Hedgehog signals (13). Microparticles have also been implicated in cirrhosis and portal hypertension, with evidence that such particles may contain the angiogenic molecule VEGF and other studies providing evidence that these particles can mediate hemodynamic disturbances associated with cirrhosis (50).…”

“…CD63 and CD81) (9 -12). Recent studies have explored potential roles for exosomes in the pathogenesis of liver inflammation, fibrosis, and portal hypertension (13). An increase in this extracellular vesicle subtype has been postulated in patients with cirrhosis (14).…”

Epithelial-mesenchymal transitions and hepatocarcinogenesis

“…Several lines of historical evidence support the concept that EMT is the biological bridge that links cirrhosis and HCC. First, markers of EMT have been detected in the blood of rodents and humans with cirrhosis (28,29). Second, various events that enhance the efficiency of cancer cell EMT predict cancer invasion, metastasis, and reduced survival in HCC patients.…”