studies

890A AASLD ABSTRACTS HEPATOLOGY, October, 2015
would use human liver, or a biologically relevant 3D in vitro
model of human liver cells grown on an extracellular matrix
(ECM) derived from healthy or cirrhotic liver tissue. Aim: The
aim of this study was to develop a rapid protocol for the decellularisation
of small samples of healthy and cirrhotic human
liver and demonstrate repopulation with cultured human liver
cell lines, namely hepatocarcinoma (HepG2) and hepatic stellate
(LX2) cells. Methods: Liver tissue cubes (125mm 3 ) were
dissected from human livers unsuitable for transplantation or
explanted liver. The decellularisation of the liver scaffolds was
completed within 3hrs (healthy) and 5hrs (cirrhotic) of agitation
in decellularization solutions i.e. detergents/enzymes. The
decellularization efficiency was determined by immunohistochemistry
for ECM components and residual DNA, scanning
electron microscopy, biomechanics, as well as DNA and ECM
protein quantification. The liver scaffolds were repopulated by
HepG2 and LX2 for up to 14 days. RESULTS: This innovative
protocol resulted in liver scaffolds with a preserved 3D structure
and ECM composition, while DNA and cellular residues
were successfully removed. The cirrhotic liver structure was
maintained after decellularization and the biomechanical properties
were remarkably different when comparing healthy liver
scaffolds with cirrhotic scaffolds. Liver scaffolds were progressively
repopulated for up to 14 days with LX2 and HepG2 cells
which showed remarkable viability, motility and proliferation
associated with remodelling effects on the surrounding ECM.
Notably, the expression of several genes and proteins involved
in liver fibrosis and cancer was different between the healthy
and cirrhotic 3D-system. CONCLUSION: This is the first report
describing an efficient protocol to completely decellularize
human liver scaffolds obtained from healthy and cirrhotic liver.
This decellularization protocol maintained the natural 3D-structure
and ECM composition and organisation of both healthy
and cirrhotic human liver tissue. This is a key advance in the
development of 3D-technologies for the study of the progression
of human liver fibrosis into cirrhosis and hepatocellular
carcinoma.
Disclosures:
Kevin Moore - Advisory Committees or Review Panels: Servier
Massimo Pinzani - Advisory Committees or Review Panels: Intercept Pharmaceutical,
Silence Therapeutic, Abbot; Consulting: UCB; Speaking and Teaching:
Gilead, BMS
The following authors have nothing to disclose: Giuseppe Mazza, Lisa Longato,
Walid Al-Akkad, Andrea Telese, Luca Urbani, Andrew R. Hall, Benjamin Robinson,
Luca Frenguelli, Oliver Willacy, Marco Curti, Domenico Tamburrino, Gabriele
Spoletini, Massimo Malago, Tu Vinh Luong, Armando E. Del Rio Hernandez,
Paolo De Coppi, Krista Rombouts
1389
Rev-erb and TGF-β Differentially Regulate Autophagy in
Hepatic Stellate Cells
Paul G. Thomes 1 , Nicole A. Feilen 1 , Jennifer H. Benbow 1 , Elizabeth
Brandon-Warner 1 , Cathy Culberson 1 , Terrence M. Donohue 2 ,
Laura W. Schrum 1 ; 1 Liver Pathobiology Laboratory, Carolinas
Medical Center, Charlotte, NC; 2 Omaha VA Medical Center &
Univ. of Nebraska Medical Center, Omaha, NE
Background: Recently, we demonstrated that ligand-activated
nuclear receptor Rev-erbα mitgates the fibrogenic phenotype of
hepatic stellate cells (HSCs) (Li et al., Hepatology, 2014). Reverbα
is also a novel regulator of macroautophagy (autophagy)
(Woldt et al., Nat Med, 2013), a crucial lysosomal degrading
system that likely provides substrates for HSC activation. Here,
we examined whether pharmacological activation of Rev-erb
with SR9009 or treatment with the pro-fibrotic cytokine, TGFβ,
each differentially modulates autophagy in activated rat
primary HSCs and in HSC or fibroblast cell lines. Methods:
We treated activated human HSC cell lines, LX2 and TWNT-4,
primary rat hepatic stellate cells (rHSCs) or mouse embryonic
fibroblast cells (3T3) with TGF-β (5 ng/ml) or Rev-erb ligand,
SR9009 (10 mM), for 24 hours. Autophagy was quantified
by protein markers and autophagosome (AV) flux by Western
blot analyses (WB) and immunohistochemistry (IHC). Results:
IHC demonstrated decreased AV following TGF-β exposure
in all cell types. Rev-erb activation with SR9009 decreased
AV in TWNT-4, 3T3 and rHSCs but not in LX2 cells. WBs
confirmed lower levels of AV marker protein, LC3II, in all TGFβ-
treated cells even after 4hr of cytokine exposure. In LX2,
TWNT-4 and rHSCs, TGF-β simultaneously decreased levels of
P62, an adaptor protein whose levels reflect the degree of AV
degradation. SR9009-treated LX2, TWNT-4 and 3T3 cells had
decreased LC3II protein levels and an insignificant decrease
in rHSCs. P62 levels were unaffected by SR9009 exposure
in all cell types. TGF-β treatment of rHSC was associated with
higher lysosome (Lys) (LAMP1) numbers and a higher co-localization
frequency of AVs with Lys. In contrast, SR9009 treatment
caused reduced Lys and AV-Lys co-localization. In rHSCs,
SR9009 but not TGF-β, decreased lysosomal cathepsin B activity.
When cells were co-treated with TGF-β and bafilomycin
(blocks AV degradation), rHSCs exhibited numerically higher
LC3II levels than cells treated with bafilomycin alone. Rat HSCs
treated with SR9009 and bafilomycin exhibited significantly
lower LC3II levels than cells treated with bafilomycin alone.
In rHSC, TGF-β and the autophagy inducers, rapamycin and
starvation decreased Rev-erbα nuclear staining. SR9009 treatment
decreased Rev-erbα cytoplasmic staining. Conclusion:
Our findings indicate that TGF-β accelerates AV degradation
and SR9009 retards AV synthesis and degradation in activated
HSCs. We propose that Rev-erbα is a transcriptional regulator
of autophagy in HSCs and that its ligand, SR9009, slows autophagic
degradation of the macromolecular substrates that fuel
fibrosis.
Disclosures:
The following authors have nothing to disclose: Paul G. Thomes, Nicole A. Feilen,
Jennifer H. Benbow, Elizabeth Brandon-Warner, Cathy Culberson, Terrence M.
Donohue, Laura W. Schrum
1390
Molecular mechanism responsible for tissue stiffness in
advanced chronic liver fibrogenesis
Takao Sakai; Department of Molecular and Clinical Pharmacology,
Institute of Translational Medicine, University of Liverpool,
Liverpool, United Kingdom
Tissue fibrosis is a part of wound-healing response that maintains
organ structure and integrity following tissue damage
and characterized by extracellular matrix (ECM) remodeling
and stiffening. However, functional contribution of tissue stiffening
to non-cancer pathogenesis remains largely unknown. In
particular, how remodeling of ECM by myofibroblasts results
in changes in mechanical tension that support the activation
of pathogenic signaling pathways remain to be elucidated.
Fibronectin is an ECM glycoprotein substantially expressed
during adult tissue repair. We have addressed the molecular
mechanism responsible for tissue stiffness in advanced chronic
liver fibrogenesis induced by carbon tetrachloride (16 weeks)
using a mouse model lacking fibronectin in the adult liver.
Fibronectin-null livers exhibited constitutively elevated local
TGF-β activity, induced more myofibroblast phenotypes, and
accumulated highly disorganized/diffuse collagenous ECM
networks composed of thinner and significantly increased number
of collagen fibrils during advanced chronic liver damage.
Consequently, fibronectin-null livers lead to more extensive liver