studies

880A AASLD ABSTRACTS HEPATOLOGY, October, 2015
Disclosures:
Scott L. Friedman - Advisory Committees or Review Panels: Pfizer Pharmaceutical;
Consulting: Conatus Pharm, Exalenz, Genfit, Exalenz Biosciences, Eli Lilly PHarmaceuticals,
Fibrogen, Boehringer Ingelheim, Nitto Corp., Immune Therapeutics,
Synageva, Roche/Genentech Pharmaceuticals, DeuteRx, Abbvie, Novartis,
RuiYi, Kinemed, Sanofi Aventis, Takeda Pharmaceuticals, Nimbus Therapeutics,
Bristol Myers Squibb, Astra Zeneca, Sandhill Medical Devices, Galmed, Northern
Biologics, Enanta Pharmaceuticals, Regado Bioscience, Raptor Pharmaceuticals,
Teva Pharmaceuticals, Zafgen Pharmaceuticals, Merck Pharmaceuticals,
Debio Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira
Pharm; Stock Shareholder: Angion Biomedica, Intercept Pharma
The following authors have nothing to disclose: Youngmin A. Lee, Luke A. Noon,
Tingfang Lee, Marie-Luise Berres, Fatemeh P. Parvin-Nejad, Kemal M. Akat, Hsin
I Chou, Varinder Athwal, M. Isabel Fiel, Ronald E. Gordon
1365
Hepatocyte- and hepatic stellate cell-specific deletion of
liver fatty acid binding protein (L-Fabp) reveals divergent,
cell-type specific roles in hepatic lipid trafficking
and fibrogenesis
Elizabeth P. Newberry 1 , Susan M. Kennedy 1 , Gianfranco Alpini 3 ,
Anping Chen 2 , Nicholas O. Davidson 1 ; 1 Washington University
School of Medicine, St Louis, MO; 2 St. Louis University, St Louis,
MO; 3 Central Texas Veterans Health Care System and Texas A&M
HSC College of Medicine, Temple, TX
Liver fatty acid binding protein (L-Fabp) modulates fatty acid
(FA) trafficking, high fat diet-induced obesity, and steatosis. We
previously showed that germline L-Fabp –/– mice have reduced
FA uptake and incorporation into complex lipid in both hepatocytes
and stellate cells (HSC), with attenuated diet-induced
hepatic steatosis and fibrosis. Since L-Fabp is expressed in
both hepatocytes and HSC, we examined its cell-specific role in
lipid trafficking and fibrotic injury. Germline L-Fabp –/– mice fed
a trans-fat, fructose (TFF) diet exhibit reduced steatosis and liver
fibrosis, with a 70% decrease in Col1a1 and αSMA mRNAs.
Hepatocyte-specific (Alb-Cre) L-Fabp deletors (Hep KO, >95%
decreased L-Fabp mRNA) exhibit reduced hepatic TG (f/f, 560
± 71mg/mg; Hep-KO, 284 ± 91; p=0.046), with decreased
liver size and expression of Col1a1 and αSMA (~50%), suggesting
that HSC activation in TFF-fed mice is linked to hepatocyte
lipid accumulation. In contrast, HSC-specific (GFAP-Cre)
L-Fabp deletion (HSC KO, ~75% reduction) did not affect liver
size, hepatic steatosis or expression of Col1a1, but αSMA
mRNA was reduced. Because GFAP Cre may target other cell
types, we verified that WT cholangiocytes express undetectable
L-Fabp. Freshly isolated HSCs from HSC-KO contain abundant
lipid droplets, in contrast to germline L-Fabp –/– HSC which are
lipid droplet depleted. Together these data indicate that TFF
diet induced fibrotic injury is linked to hepatocyte TG content
and suggest that lipid trafficking to HSCs may reflect FA compartmentalization
in hepatocytes. In a distinct model of liver
injury, carbon tetrachloride (CCl 4
) administration produced
worse injury in L-Fabp –/– mice vs C57BL/6J controls (ALT WT,
666 ± 194 IU/L; KO, 1247 ± 179), with increased hepatocyte
proliferation and enhanced expression of fibrogenic genes,
yet no difference in fibrosis (WT, 2.6 ± 0.5%; KO, 2.4 ± 0.3,
n=6). We then used HSC- and Hep- KOs to understand how
L-Fabp deletion exacerbates CCl 4
-induced liver injury. CCL 4
-
treated HSC KO mice exhibited higher serum ALT (f/f, 733
± 306 IU/L; HSC-KO, 1450 ± 270, p=0.09) and hepatocyte
proliferation (f/f, 7.2 ± 3.1 % BrdU+; HSC-KO 16.6 ± 1.6,
p=0.02) with ~3-fold increased Col1a1 and αSMA expression.
In contrast, CCl 4
-treated Hep KO mice exhibited no difference
in ALT (548 ± 186 IU/L) or hepatocyte proliferation
versus controls. Together these data indicate that HSC L-Fabp
expression plays a protective role against CCl 4
injury, whereas
hepatocyte L-Fabp promotes TFF diet-induced steatotic injury.
Furthermore, the findings point to an unanticipated, cell specific
role for L-Fabp in high fat diet induced versus toxin-mediated
liver injury.
Disclosures:
The following authors have nothing to disclose: Elizabeth P. Newberry, Susan M.
Kennedy, Gianfranco Alpini, Anping Chen, Nicholas O. Davidson
1366
Fibrous network composition and cell heterogeneity
modulate long-range force transmission by cells in
matrices
Robert N. Kent, Jude D. Han, Rebecca G. Wells; Gastroenterology,
University of Pennsylvania, Philadelphia, PA
Long-range force transmission is implicated in the large-scale
architectural rearrangements occurring in fibrotic diseases like
pulmonary fibrosis and liver cirrhosis. It was previously shown
that matrix organized in a fibrous network is required and
that collagen becomes aligned and compacted as part of this
process. We hypothesized that the characteristics of the fibrous
network as well as the heterogeneity of the cell population
modulate long-range force transmission between cells. Fibroblasts
(murine 3T3) were cultured as spheroids by suspension
in a hanging droplet over 5 days. These were seeded in pairs
500-1000 μm apart on thick gels of either denatured collagen
(gelatin) or type I collagen (Col1) containing varying concentrations
of type III collagen (Col3) or plasma fibronectin (pFN).
Heterotypic spheroids containing varying ratios of primary rat
hepatic stellate cells (HSCs) and hepatocytes were cultured in
hanging droplets for 3 days and seeded on Col1 gels. Once
seeded, spheroids were incubated or imaged via time-lapse
microscopy for 24 hours and then fixed. Collagen alignment
and compaction were visualized using second harmonic generation
imaging, and the extent of alignment was quantified by
calculating the anisotropy index in ImageJ (NIH). Gels without
spheroids showed minimal collagen organization or alignment
by second harmonic generation imaging. Col1 gels with spheroids
showed marked alignment, contraction, and compaction
of collagen along the axis between spheroids, with migration of
cells along the compacted fibrils. Spheroids of 3T3 fibroblasts
seeded on gelatin substrates showed minimal organization,
suggesting that collagen cross-linking is required for cell-mediated
network reorganization. On substrates consisting of 60%
Col1 and 40% Col3, 3T3 spheroids generated significantly
less fiber alignment when compared to spheroids on pure Col1
substrates (p < 0.01). Col1 gels containing pFN (10% v/v),
however, yielded a significant increase in alignment relative to
pure Col1 (p < 0.05). Spheroids of primary HSCs reorganized
the fibrous network such that the anisotropy index was significantly
larger than the no-spheroid control (p < 0.05). The inclusion
of hepatocytes in ratios of 1:20 and 1:1 hepatocyte:HSC
yielded even greater network anisotropies than spheroids of
HSCs alone (p < 0.01, 0.05). Collagen cross-linking, Col3 and
pFN content, and heterogeneity of cell populations modulate
long-range force transmission in fibrous networks. Determining
the role of matrix cross-linking and cell heterogeneity in matrix
reorganization may contribute to understanding the underlying
mechanisms driving bridging in liver cirrhosis.
Disclosures:
The following authors have nothing to disclose: Robert N. Kent, Jude D. Han,
Rebecca G. Wells