studies

896A AASLD ABSTRACTS HEPATOLOGY, October, 2015
1403
Modulation of Extracellular Matrix Remodeling by Activation
of the Aryl Hydrocarbon Receptor
Cheri L. Lamb 1,2 , Kristen A. Mitchell 1,2 ; 1 Biological Sciences, Boise
State University, Boise, ID; 2 Biomolecular Sciences Ph.D. Program,
Boise State University, Boise, ID
The aryl hydrocarbon receptor (AhR) is a soluble, ligand-activated
transcription factor involved in developmental processes,
xenobiotic metabolism, and adaptation to environmental stress.
Recent evidence indicates that this receptor may also contribute
to wound healing processes. We recently found that exposure
to the potent AhR agonist 2,3,7,8 tetrachlorodibenzo-p-dioxin
(TCDD) increases activation of hepatic stellate cells, which
contribute to hepatic wound healing and fibrosis through the
deposition of extracellular matrix material, namely collagen
type I. The goal of the present study was to determine how
TCDD treatment impacts matrix deposition and remodeling
activities during experimental liver fibrosis elicited by carbon
tetrachloride (CCl 4
). Male C57Bl/6 mice were treated with
CCl 4
(5 ml/kg diluted 1:10 in corn oil) twice a week for eight
weeks. During the final two weeks, mice were treated with
TCDD (20 μg/kg by gavage) or vehicle (peanut oil). Collagen
deposition was measured by cytohistological staining and
evaluated by polarized microscopy. Levels of matrix enzymes
and associated regulatory proteins were measured by qPCR,
and activity was assessed by in situ zymography. Results indicate
that TCDD treatment increased Col1a1 mRNA levels and
altered the pattern of collagen deposition in the fibrotic septa.
Exposure to TCDD also increased in situ collagenase activity
in the fibrotic liver with a concomitant 4-fold increase in matrix
metalloproteinase-13 mRNA levels. Inhibitors of matrix metalloproteinases,
namely PAI-1 and TIMP-1, were increased in
TCDD-treated mice, which could reflect direct AhR transcriptional
activity or else a compensatory response to elevated
collagenase activity. Collectively, these results indicate that
TCDD-induced AhR activation not only increases fibrogenesis,
but also perturbs extracellular matrix remodeling activities in
the fibrotic liver. Hence, these findings implicate a previously
unidentified role for the AhR in modulating wound healing
responses in the liver and may provide a basis for designing
therapeutic strategies to limit fibrosis by modulating AhR activation.
Disclosures:
The following authors have nothing to disclose: Cheri L. Lamb, Kristen A. Mitchell
1404
MicroRNA profiling of circulating exosomes in HBV cirrhosis
patients following anti-HBV therapy
Min Cong 1 , Li Chen 2 , Tianhui Liu 1 , Ping Wang 1 , Jidong Jia 1 , David
Brigstock 2 , Hong You 1 ; 1 Liver Research Center, Beijing Friendship
Hospital, Beijing, China; 2 The research Institute at Nationwide
Children’s Hospital, Columbus, OH
Aim: The objective of our study is to establish serum exosomal
microRNA (miR) signatures to predict progression and reversion
of fibrosis in hepatitis B (HBV)–induced cirrhosis undergoing
anti-viral therapy. Methods: Twelve HBV patients with
compensated cirrhosis were selected based on their cirrhosis
regression (decreased Fibroscan values of > 5kPa) or progression
(increased or stable Fibroscan values) after treatment for 6
months with Entecavir (0.5mg/day oral), respectively. Twelve
matched healthy blood donors were used as a reference control
group. Serum (0.5ml) from the HBV patients, before or
after therapy, or from the controls was individually processed
to purify exosomes from which small RNA was then isolated.
Samples in each group were pooled and analyzed using a
Human miRNome PCR array to profile the1066 most abundantly
expressed human miR sequences using SYBR Green real
time PCR. Circulating exosomalmiRs were isolated from 8 other
compensated cirrhosis HBV patients who presented with cirrhosis
regression or progression to validate the change of selected
exosomalmiRs by qRT-PCR. Results: Compared with control
subjects, 59 out of 1066 miRs from 12 regressed cirrhosis
HBV patients were up- or down-regulated at least 15-fold in the
cirrhotic patients, with 46 out of 59 miRs showing correction
(to more healthy) after therapy. Among the miRs identified in
this unbiased analysis were miR-7, -15b, -16, -22, -26a, -27b,
-29, -92b, -101, -183, -191, -196a, -200a, -370, -378a and
-936 and let-7a-g which have been reported in the literature to
be fibrosis-related in the liver or other organ systems. Analysis
of exosomalmiRs from 12 progressed cirrhosis HBV patients
showed that these miRs were less corrected as compared to
the regressed patients and that different miRs served to discriminate
these patients from the healthy controls. qRT-PCR analysis
of exosomalmiRs from 8 cirrhosis HBV patients, before or after
therapy, showed that miR-16,-20a,-25,-148a,-223,-320d were
significantly decreased while miR-126,-191 were significantly
increased in progressed patients. Conclusions: These data
reveal that the circulating exosomalmiR content varies according
to the stage of HBV fibrosis and that exosomalmiRs show
quantitative and qualitative changes that reflect the patients’
clinical course of fibrosis regression or progression after anti-viral
treatment. Since circulating exosomalmiRs change dynamically
with disease status, these findings highlight the possibility
of identifying discriminatory miR signatures that are diagnostic
or predictive for fibrosis and that can be obtained longitudinally
and non-invasively in individual patients.
Disclosures:
Jidong Jia - Consulting: BMS, MSD, Roche
David Brigstock - Stock Shareholder: FibroGen
The following authors have nothing to disclose: Min Cong, Li Chen, Tianhui Liu,
Ping Wang, Hong You
1405
Identification of Axon guidance signaling pathway
mediators in hepatic stellate cells and liver fibrogenesis
Jinsheng Guo 1 , David Zhang 2 , Yujing Wu 1 , Daisuke Hasegawa 2 ,
Scott L. Friedman 1,2 ; 1 Division of Digestive Diseases, Zhong Shan
Hospital, Fu Dan University, Shanghai, China; 2 Division of Liver
Diseases, Icahn School of Medicine at Mount Sinai, New York, NY
Background&Aims: Hepatic stellate cells (HSC) have many
features of neural cells including expression of glial fibrillary
protein, nestin and neurotrophin receptors. We recently used
an informatics based approach to identify novel transcripts
expressed by stellate cells encoding cell surface and secreted
proteins (Zhang DY et al, Gut, 2015, in press). Among these,
Robo2 is a cell surface molecule implicated in axon guidance.
The aim of our study was to identify the novel signaling pathway
and key molecules in liver fibrogenesis by dynamic network
analysis of liver transcriptomes. Methods&Results: Two
experimental models of hepatic fibrosis were employed by
intraperitoneally injection of diethylnitrosamine (DEN, 10mg/
kg/week) in mice for 14 weeks, or by intraperitoneal injections
of carbon tetrachloride (CCl4, 0.9ml/kg/biw) for four weeks.
In the DMN model, the liver samples were collected at 0, 6, 8
and 14 weeks post DEN treatment for RNA sequencing. The
transcriptomic data associated with development of liver fibrosis
(i.e., injury, early fibrotic, late fibrotic or cirrhotic stages)
were applied to time-series analysis, followed by network
and pathway analyses. Among markedly up-regulated genes