studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 879A
1363
Mast cells interact with proliferating cholangiocytes to
activate hepatic stellate cells and promote fibrosis via
TGF-β1 signaling during cholestatic injury
Lindsey Kennedy 2 , Laura Hargrove 1 , Jennifer Owens 2 , Heather
L. Francis 2,1 ; 1 Scott and White Memorial Hospital, Temple, TX;
2 Research, Central Texas Veteran’s Health Care System, Temple,
TX
Background: Hepatic fibrosis is marked by activation of hepatic
stellate cells (HSCs) and the release of TGF-β1. Cholestatic
injury is a precursor to liver fibrosis and, following injury cholangiocytes
acquire a neuroendocrine phenotype and interact
with HSCs to promote fibrosis. Mast cells (MCs) infiltrate the
liver following injury and release pro-fibrogenic factors including
histamine that increases biliary proliferation and fibrosis.
We tested the hypotheses that (i) MCs induce biliary proliferation,
HSC activation and promote fibrosis and (ii) inhibition of
MC-derived histamine decreases fibrosis via TGF-β1. Methods:
To demonstrate that MCs promote fibrosis, wild-type (WT) and
HDC -/- mice (characterized by lower MC count) were subjected
to sham surgery or bile duct ligation (BDL) and injected with
cultured MCs via tail vein. Further, WT and MDR2 -/- mice (a
model of Primary Sclerosing Cholangitis characterized by
increased MC count) were treated with cromolyn sodium to
block MC-derived histamine. We collected liver blocks, serum,
cholangiocytes and biliary supernatants. Biliary mass and proliferation
were evaluated by immunohistochemistry for CK-19
and PCNA, respectively. Fibrosis was evaluated in tissue sections
by Sirius Red/Fast Green Collagen staining and by qPCR
in total liver and cholangiocytes for α-SMA, fibronectin, collagen
type 1a and TGF-β1. HSC activation was evaluated by
qPCR in total liver and immunofluorescent staining in tissues
for synaptophysin 9 (SYP9). TGF-β1 secretion was measured in
serum from all groups. Downstream from TGFβ-1, we measured
SMAD2 and SMAD3 by qPCR and western blotting in total
liver and cholangiocytes. In vitro, cultured MCs were transfected
with HDC shRNA to decrease histamine secretion and
subsequently co-cultured with cholangiocytes or HSCs prior to
measuring fibrosis markers, proliferation and TGF-β1 secretion.
Results: Injected mast cells were found in close proximity to proliferating
bile ducts. Biliary proliferation, fibrosis and HSC activation
were increased in HDC -/- and BDL HDC -/- mice following
MC injection. Inhibition of MC-derived histamine decreased
biliary proliferation, fibrosis, TGF-β1 secretion and SMAD2/3
expression in MDR2 -/- mice. In vitro, knockdown of MC HDC
decreased (i) MC TGF-β1 secretion, (ii) biliary proliferation and
fibrotic marker expression and (iii) HSC activation and TGF-β1
secretion. Conclusion: MCs are recruited to proliferating cholangiocytes
and promote HSC activation increasing fibrosis via
TGF-β1/SMAD signaling. Inhibition of MC-derived histamine
decreases fibrosis and regulation of MC mediators may be a
therapeutic target for fibrosis.
Disclosures:
The following authors have nothing to disclose: Lindsey Kennedy, Laura Hargrove,
Jennifer Owens, Heather L. Francis
1364
Hepatocyte autophagy impairment promotes ductular
reaction and fibrogenesis by hepatic stellate cells
Youngmin A. Lee 1 , Luke A. Noon 1,2 , Tingfang Lee 1 , Marie-Luise
Berres 3,6 , Fatemeh P. Parvin-Nejad 1 , Kemal M. Akat 4 , Hsin I
Chou 1 , Varinder Athwal 1,7 , M. Isabel Fiel 5 , Ronald E. Gordon 5 ,
Scott L. Friedman 1 ; 1 Liver Diseases, Icahn School of Medicine at
Mount Sinai, New York, NY; 2 Centro de Investigación Principe
Felipe (CIPF), Valencia, Spain; 3 Department of Internal Medicine
III, University Hospital, RWTH Aachen, Aachen, Germany; 4 Laboratory
of RNA Molecular Biology, HHMI/Rockefeller University,
New York, NY; 5 Department for Pathology, Icahn School of Medicine
at Mount Sinai, New York, NY; 6 Department of Oncological
Sciences, Icahn School of Medicine at Mount Sinai, New York,
NY; 7 Institute of Human Development, Faculty of Medicine and
Human Sciences, University of Manchester, Manchester, United
Kingdom
Background Ductular reaction (DR), an expansion of ductular
progenitor-like cells, is often correlated with fibrosis in chronic
liver disease and fulminant liver injury. Murine models in which
autophagy, a homeostatic lysosomal pathway, is abrogated
in hepatocytes by deletion of the autophagy related protein 7
(Atg7) display a prominent DR with marked elevation of liver
transaminases, steatohepatitis and fibrosis, with eventual tumorigenesis.
Although hepatic stellate cells (HSCs) are associated
with the DR, their interactions have not been well characterized
and their relative contribution to fibrosis is uncertain. Our
aim was to determine the contribution of ductular and HSCs
to fibrosis when hepatocyte autophagy is impaired. Methods:
Olig1-Cre:Atg7 F/F mice, which have selective hepatocyte
depletion of the autophagy protein Atg7, were analyzed by
immunohistochemistry (IHC), qRT-PCR and microarray for fibrogenic
markers. Primary HSCs and ductular cells were isolated
following collagenase perfusion and gradient purification.
Enrichment of respective cell populations was confirmed by
flow analysis and IHC. Gene expression was then analyzed by
qRT-PCR. To identify fibrogenic cells in vivo, Olig1-Cre:Atg7 F/F
mice were crossed to transgenic Col1a1-GFP mice, in which
GFP expression is regulated by the Collagen1a1 promoter.
Livers of Tg(Col1-GFP)cKO(Olig1-Cre:Atg7 F/F ) were analyzed
by IHC for GFP as well as markers for ductular cells (EpCAM,
CD133) and HSCs (desmin). Results: Olig1-Cre:Atg7 f/f mice
develop extensive ductular reaction (Epcam + , CD133 + by IHC)
and marked spontaneous fibrosis at 3 months, as assessed by
sirius red staining (8.4 ± 2.4 vs 1.1±0.2 % fibrotic tissue area
in controls), with significant fibrogenic gene induction in whole
liver RNA based on qRT-PCR and array. Strikingly, collagen
fibers were closely aligned with ductular cells (EpCAM + , Ck7 + )
that were also intimately associated with HSCs (desmin + ).
HSCs and ductular cells isolated from Olig1-Cre:Atg7 F/F both
expressed aSMA, a marker of activated HSCs, and Col1,
whereas none was expressed in control livers. In transgenic
Col1a1-GFP reporter mice with hepatocyte autophagy loss,
GFP expression was greater in desmin positive cells than in
cells expressing progenitor markers (EpCAM, CD133), establishing
HSCs as the primary fibrogenic cells. Conclusion:
Hepatic fibrosis is a prominent and novel feature of hepatocyte
Atg7 KO mice. HSCs intimately associated with ductular cells
are the main fibrogenic cell population. These findings help
clarify the basis of fibrogenesis associated with the DR and
establish an experimental model for elucidating cell-cell interactions
in this response.