studies

304A AASLD ABSTRACTS HEPATOLOGY, October, 2015
188
Splenectomy attenuates advanced liver fibrosis stimulating
hepatic accumulation of anti-fibrogenic monocytes/
macrophages
Yuji Iimuro 1,2 , Akito Yada 1 , Toshihiro Okada 1 , Naoki Uyama 1 ,
Jiro Fujimoto 1 ; 1 Department of Surgery, Hyogo College of Medicine,
Hyogo, Japan; 2 Surgery, Nirasaki Municipal Hospital, Nirasaki,
Japan
Splenectomy (SP), which is performed in cirrhotic patients with
hypersplenism, has been reported to improve liver function;
however the underlying mechanism remains obscure. The AIM
of the present study was to investigate the mechanism using
a murine model and human liver tissues. Methods: C57BL/6
male mice were allowed to drink water including thioacetamide
(TAA: 300 mg/l) ad libitum for 32 weeks. After SP at
32 weeks, mice were sacrificed on days 1, 7, and 28, respectively,
while TAA-administration was continued. Perioperative
changes in peripheral blood and liver tissues were analyzed.
In cirrhotic patients with hepatocellular carcinoma (HCC), who
underwent laparoscopic SP in advance, non-tumorous liver
tissues obtained from resected specimen were histologically
analyzed. Results: TAA treatment of mice for 32 weeks reproducibly
achieved advanced liver fibrosis with splenomegaly,
thrombocytopenia, and leukocytopenia, well representing
the compensated liver cirrhosis in humans. After SP, thrombocytopenia
and leukocytopenia were quickly improved, and
liver fibrosis was obviously attenuated. Among the leukocytes,
monocytes/macrophages were selectively increased in peripheral
blood, as well as in the liver. Meanwhile, progenitor-like
cells expressing CK-19, EpCAM, or CD-133 appeared along
the fibrous scar in the liver after TAA treatment, and gradually
disappeared after SP. Monocytes/macrophages accumulated
in the liver, most of which were negative for Ly-6C, existed
adjacent to the hepatic progenitor-like cells. qRT-PCR indicated
increased canonical Wnt and decreased Notch signals in
the liver, and Wnt2 protein expression in monocytes/macrophages
was confirmed. A significant amount of β-catenin accumulated
in the adjacent progenitor-like cells, and Ki67-positive
relatively small hepatic cells were significantly increased. Protein
expression of MMP-9, to which Ly-6G-positive neutrophils
contributed, was also increased in the liver after SP. In cirrhotic
patients with HCC, thrombocytopenia and lekocytopenia were
quickly attenuated after SP, and the percentage of monocytes
in peripheral blood was selectively increased. In the their liver
tissues, remarkable accumulation of round- or oval-shaped macrophages/monocytes
positive for CD163 was detected after
SP, and they existed closely to the CK19-positive cells, which
spread out from the ductular reactions, suggesting interaction
between these cells. Conclusions: The hepatic accumulation of
monocytes/macrophages, the reduction of fibrosis, and the
gradual disappearance or expansion of hepatic progenitor-like
cells, possibly play significant roles in the tissue remodeling
process in cirrhotic livers after SP.
Disclosures:
The following authors have nothing to disclose: Yuji Iimuro, Akito Yada, Toshihiro
Okada, Naoki Uyama, Jiro Fujimoto
189
The role of Mesothelin in the pathogenesis of cholestatic
liver fibrosis
Yukinori Koyama 2,1 , Ping Wang 2,1 , David A. Brenner 1 , Tatiana
Kisseleva 2 ; 1 Medicine, University of Californiam, San Diego, La
Jolla, CA; 2 Surgery, University of California, San Diego, CA
Background: There are no curative treatments for patients
with the cholestatic liver diseases, primary sclerosing cholangitis
(PSC), and primary biliary cirrhosis (PBC). Although the
etiology of PSC and PBC remains unclear, activated portal
fibroblasts (aPFS) have been implicated in pathogenesis of
cholestatic clinical and experimental liver fibrosis. We have
identified that mesothelin (Msln) is a specific marker for portal
fibroblasts. Aim: To examine the role of Msln in cholestatic liver
fibrosis in mice. Methods: Using Msln as a specific marker of
aPFs, we investigated the development of BDL-induced liver
fibrosis in mice devoid of aPFs (Msln DTA mice) that were generated
by expression of Diphtheria toxin-a (DTA) in Msln + cells
(by crossing the Msln ER-Cre and Rosa26 flox-Stop-flox-DTA mice).
Next, the role of Msln in cholestatic liver fibrosis was examined
in Msln knockout mice (Msln -/- mice). To explore the role Msln
in TGF-β signaling, we generated transgenic Msln DSmad4 mice
in which Smad4 was specifically deleted in Msln + aPFs. Wt
littermates served as controls. All mice were subjected to bile
duct ligation (BDL), and livers were analysed for development
of liver fibrosis. aPFs from wt and Msln -/- mice were isolated
and analysed by RNA-Seq. Finally, to examine if Msln can be
a new therapeutic target for cholestatic liver fibrosis, BDL wt
mice were treated with anti-Msln Ab (5 mg, 10 mg / mouse).
Results: aPF-ablated Msln DTA mice developed 50% less fibrosis
in response to BDL than wt mice, demonstrating the requirement
of aPFs for cholestatic liver fibrosis. Msln-/- mice had
less fibrosis in response to BDL (5 and 17 days), but this effect
was not observed in carbontetrachloride (CCl 4
)-treated mice,
demonstrating a role for Msln in cholestatic but not hepatotoxic
induced liver fibrosis. Msln -/- aPFs exhibit a defect in migration
and proliferation by modulating TGF-β1 signaling, as demonstrated
by reduced number of cells populating the scratch area,
and decreased expression of Ki-67 by Msln -/- aPFs. Furthermore,
we found that expression of TGF-β1 target genes, such
as TGF-β2, PAI-1 and Activin1, were strongly downregulated
in Msln -/- aPFs compared with wt aPFs, and were associated
with reduced phosphorylation of Smad2 when stimulated with
TGF-β1. The Msln DSmad4 mice developed less fibrosis. Administration
of anti-Msln antibody to BDL-injured mice injury attenuated
liver fibrosis in a dose dependent manner (compared to
IgG control). Conclusion: aPFs and in particular the expression
of Msln in aPFs play important roles in the pathogenesis of
cholestatic liver fibrosis. Msln might become a novel target for
treatment of cholestatic liver fibrosis.
Disclosures:
The following authors have nothing to disclose: Yukinori Koyama, Ping Wang,
David A. Brenner, Tatiana Kisseleva