studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 875A
1354
Myofibroblastic conversion of portal fibroblasts and
mesothelial cells isolated from adult mouse liver
Ingrid A. Lua, Kinji Asahina; University of Southern California,
Department of Pathology, Keck Scool of Medicine, Los Angeles,
CA
Purpose: Hepatic stellate cells (HSCs) are believed to be the
major source of myofibroblasts in liver fibrosis; however, other
cellular sources, such as portal fibroblasts (PFs) in the portal
triad and mesothelial cells (MCs) on the liver surface, have also
been suggested. It is unknown how these cells contribute to
fibrogenesis due to insufficient availability of markers and isolation
methods. Methods: We isolated HSCs, PFs and MCs from
collagen1a1 (Col1a1)-GFP transgenic mouse livers by fluorescence-activated
cell sorting (FACS) and identified their markers
by microarray analysis. Primary HSCs, PFs and MCs were
cultured in the presence of TGF-β1 or PDGF-BB to examine their
myofibroblastic conversion and proliferation. Results: Immunohistochemistry
showed that Col1a1-GFP mouse livers broadly
express GFP in quiescent HSCs, PFs, and MCs. Combining
vitamin A (VitA) lipid autofluorescence with GFP expression,
we separated the VitA+ HSCs and VitA-GFP+ population from
the Col1a1-GFP livers through FACS. We further subtracted
glycoprotein M6A (GPM6A)+ MCs from the heterogeneous
VitA-GFP+ population and obtained a PF-enriched fraction as
VitA-GFP+GPM6A- cells. Microarray analysis identified selective
expression of Reelin in HSCs. PFs exclusively expressed
Ectonucleoside triphosphate diphosphohydrolase-2 (ENTPD2/
NTPDase2). Isolated PFs exhibited fibroblastic morphology
without storing VitA lipids in culture. MCs showed a round
morphology and expressed CD200, mesothelin, podoplanin,
and uroplakin 1b. HSCs, PFs, and MCs that were isolated from
normal Col1a1-GFP livers transformed into myofibroblasts that
expressed α-smooth muscle actin induced by TGF-β1 in culture.
TGF-β1 suppressed Cyclin D mRNA expression in PFs but not in
HSCs and MCs. By contrast, PDGF-BB induced Cyclin D mRNA
only in HSCs. Conclusion: HSCs, PFs, and MCs have the potential
to differentiate into myofibroblasts, and their proliferation is
regulated differently by TGF-β1 and PDGF-BB.
Disclosures:
The following authors have nothing to disclose: Ingrid A. Lua, Kinji Asahina
1355
Selective disruption of dynamin GTPase activity in HSC
increases murine fibrogenesis in vivo and promotes HSC
activation in vitro
Qian Ding 1 , Ruisi Wang 1 , Thiago de Assuncao 1 , Meng Yin 2 ,
Sheng Cao 1 , Usman Yaqoob 1 , Richard Ehman 2 , Robert C. Huebert
1 , Vijay Shah 1 ; 1 Gastroenterology Research Unit, Mayo Clinic,
Rochester, MN; 2 Department of Radiology, Mayo Clinic, Rochester,
MN
Background: Dynamin-2 (Dyn2) is a large GTPase responsible
for endocytosis. A point mutation of Dyn2 at lysine 44 to
alanine disrupts dynamin GTPase activity (Dyn2K44A). Prior
studies using a mouse we generated that conferred Dyn2K44A
expression selectively in endothelial cells impaired angiogenesis
in vivo by disrupting receptor tyrosine kinase endocytosis
and signaling. We hypothesized that expression of Dyn2K44A
in hepatic stellate cells (HSC) would similarly disrupt tyrosine
kinase dependent HSC activation and fibrogenesis in vivo.
Methods and Results: Dyn2K44A fl/fl mice were crossed with
Collagen 1-Cre (Col1 Cre ) mice to generate offspring with HSC
selective expression of Dyn2K44A (Col1 Cre /Dyn2K44A fl/fl ).
Primary HSC isolates showed Dyn2K44A expression by realtime
PCR. Col1 Cre /Dyn2K44A fl/fl mice and littermate controls
were subjected to carbon tetrachloride (CCl 4
) or olive oil vehicle
for 6 weeks, or bile duct ligation (BDL) or sham operation
for 3 weeks to induce fibrosis and then sacrificed. Contrary
to our hypothesis, immunofluorescence for α-smooth muscle
actin (α-SMA) and fibronectin (FN), and sirius red staining from
fixed liver tissues was significantly greater in response to both
BDL and CCl 4
in Col1 Cre /Dyn2K44A fl/fl mice compared with
littermate controls (~1.5-fold; p