studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 613A
On the other hand, in PC2-defective cells STIM-1 couples with
a Ca 2+ -inhibitable Adenylyl Cyclase (ACs) and stimulates the
store-operated production of cAMP (SOcAMP), PKA-dependent
activation of the ERK1/2 and increase in VEGF production
by cystic cells. This mechanism plays a key role in promoting
liver cyst growth in PLC. Two Ca 2+ -inhibitable ACs (AC6 and
AC5) are expressed in cholangiocytes and have similar properties,
however the molecular identity of the AC responsible for
inappropriate cAMP production in PLC is unknown. Results:
PC2-conditional KO mouse (Pkd2 flox/- ;pCxreER TM ) (Pkd2cKO)
were crossed with AC6 -/- mouse to generate double Pkd2/
AC6KO mice. Successful deletion was confirmed by RT/PCR.
Mice were sacrificed eight weeks after induction of PC2 excision
and cystic area was assessed by K19 staining and computer-assisted
morphometric analysis. However, Pkd2/AC6KO
mice did not show a reduction of the liver cystic area as respect
to Pkd2cKO mice. Moreover, in cystic cholangiocytes isolated
from Pkd2/AC6KO mice, intracellular levels of cAMP generated
after an acute ER [Ca 2+ ] depletion (TPEN 1mM) were not
different from those measured in cholangiocytes from Pkd2cKO
mice, suggesting that in absence of AC6, the AC5 isoform, may
be activated. In fact, inhibition of AC5 by two different inhibitors
(NKY90 and SQ 22,536) significantly reduced cAMP
following ER [Ca 2+ ] depletion. Consistent with these data, in
vivo treatment of Pkd2/AC6KO with SQ 22,536 ((300 mg/Kg
day, I.P) caused a significant reduction in liver cystic area was
significantly reduced, along with the liver/body weight ratio.
In conclusion, these data indicate that AC5 is the Ca2+ inhibitable
AC responsible for cAMP production by PC2-defective
cells in condition of low cellular and ER [Ca 2+ ] and the mechanisms
is of pathophysiological relevance. Inhibition of cAMP
production by somatostatin analogs has been shown to be of
therapeutic value in patients with PLC. Our study indicate that
pharmacologic inhibition of AC5 may represent a more specific
target to reduce of cyst growth in polycystic liver diseases.
Disclosures:
The following authors have nothing to disclose: Carlo Spirli, Ambra Villani, Valeria
Mariotti, Luca Fabris, Romina Fiorotto, Mario Strazzabosco
812
Cholestatic-induced biliary proliferation and fibrosis
are decreased in mice lacking mast cells: an innovative
study using C-kit knockout mice
Laura Hargrove 1 , Lindsey Kennedy 2 , Jennifer Owens 2 , Heather
L. Francis 2,1 ; 1 Scott and White Memorial Hospital, Temple, TX;
2 Research, Central Texas Veterans Health Care System, Temple, TX
Background: Cholestatic liver injury is marked by increased
biliary proliferation. Bile duct ligation (BDL) induces large, but
not small, cholangiocyte proliferation coupled with increased
fibrosis. Mast cells (MCs) are inflammatory cells that, once
activated, release histamine (HA). Our previous studies have
demonstrated that HA increases vascular endothelial growth
factor (VEGF) expression and knockdown of histidine decarboxylase
(HDC (synthesizes histamine)) decreases biliary VEGF
expression/secretion. We have demonstrated that MCs infiltrate
the liver following BDL increasing intrahepatic bile duct
mass (IBDM) and fibrosis. Inhibition of MC-derived HA using
cromolyn sodium decreases these parameters suggesting that
MCs contribute to biliary proliferation and fibrosis. Our aim
was to evaluate the effects of BDL on biliary proliferation and
fibrosis in MC deficient mice. Methods: WT and C-kit -/- mice
(MC deficient) were subjected to sham or BDL for 3 days before
collecting serum, liver blocks and pure cholangiocytes. IBDM
and proliferation were evaluated in sections by CK-19 and
PCNA immunohistochemistry, respectively. In total liver (TL)
and cholangiocytes we evaluated PCNA expression by qPCR
and western blotting. Apoptosis was measured by gene expression
for Bax and BCL-2 and TUNEL staining in tissues. Fibrosis
was detected by Sirius Red/Fast Green staining in tissues and
by qPCR for α-SMA, fibronectin, collagen type-1a and TGFβ1
in TL. In cholangiocytes and TL we measured HDC and
VEGF-A/C, gene expression and secretion by EIA in serum
and cholangiocyte supernatants. To determine if MC-derived
HA regulates biliary proliferation and fibrosis in vitro, cultured
MCs were transfected with HDC shRNA prior to co-culture with
murine cholangiocytes. Biliary proliferation was measured by
MTS, and VEGF expression and fibrosis marker expression by
qPCR. Results: In BDL C-kit -/- mice there was enhanced ductular
reaction with increased small IBDM and decreased large IBDM
compared to BDL WT. Proliferation and liver fibrosis were
decreased in BDL C-kit -/- mice compared to BDL WT coupled
with enhanced apoptosis. HDC and VEGF-A/C expression was
decreased in BDL C-kit -/- mice as was HA and VEGF secretion
in serum and supernatants compared to BDL WT. In vitro,
knockdown of MC-HDC expression decreased biliary proliferation,
VEGF expression and fibrosis suggesting MC-derived
HA contributes to biliary proliferation and fibrosis. Conclusion:
BDL-induced biliary proliferation and fibrosis are decreased in
mice lacking mast cells via the HDC/HA/VEGF axis. Our studies
support the novel concept that hepatic mast cells are critical
to biliary response following injury.
Disclosures:
The following authors have nothing to disclose: Laura Hargrove, Lindsey Kennedy,
Jennifer Owens, Heather L. Francis
813
YAP Reduces Liver Parenchymal Damage by Promoting
Bile Duct Capacity in Cholestatic Injury
Quy P. Nguyen 1 , Nan Wu 2 , Tianhao Zhou 2 , Haibo Bai 1 ; 1 BaylorScott&White
Hospital, Temple, TX; 2 Texas A&M University College
of Medicine, Temple, TX
Background&Aims: YAP is highly expressed in cholangiocytes.
YAP protein levels are further increased in cholangiocytes after
bile duct ligation (BDL). However, the physiological role of
biliary YAP in maintaining biliary homeostasis and in response
to cholestatic injury is not clear due to lacking efficient methods
to ablate YAP in cholangiocytes. Methods: We found that overexpression
of YAP in hepatocytes induces a compensatory loss
of YAP in cholangiocytes. Moreover, even suppressing YAP
activity with transgenic Vgl4, a YAP competitor, is not able to
rescue the YAP deficiency in cholangiocytes. Because Yap/
Vgl4 double transgenic mice (Yap/Vgl4 DTg) have normal
liver function, we use it as model mice to study the function of
YAP in cholangiocytes. Sham or BDL was performed with these
groups of mice. Two weeks later, liver histology, double immunofluorescence
staining (E-cadherin and CK19), immunohistochemistry
staining (CK19), Sirius red staining were compared
between control and Yap/Vgl4 DTg groups. Primary cholangiocytes
were isolated from CK19-CreER; Yap flox/flox mice and
cultured in vitro. Yap deletion was induced through addion of
4-OH tamoxifen. Immunofluorescence staining of E-cadherin
was compared between control and Yap KO cholangiocytes.
YAP expression in bile ducts of the Mdr2 -/- mouse model of
primary sclerosing cholangities was also studied by immunohistochemistry.
Results: Bile infarcts and fibrosis were more
severe in Yap/Vgl4 DTg mice compared to their WT littermate
controls. CK19 staining revealed bile ducts are more dilated
in WT controls than in Yap/Vgl4 DTg livers. E-cadherin staining
revealed more well formed E-cadherin junction between
cholangiocytes in Yap/Vgl4 DTg mice. E-cadherin junctions