studies

298A AASLD ABSTRACTS HEPATOLOGY, October, 2015
175
Transcriptomic Analysis Of Human Hepatocytes In
3-Dimensional Cultures With Maintained Perfusion And
Transport Offers Insights Into The Pharmacotoxicology
Of Clinically Relevant Concentrations Of Obeticholic
Acid.
Arun J. Sanyal 2 , Robert Figler 1 , Brett R. Blackman 1 , Svetlana
Marukian 1 , Sol Collado 1 , Mark Lawson 1 , Aaron J. Mackey 1 ,
David Manka 1 , Brian R. Wamhoff 1 , Ajit Dash 1 ; 1 HemoShear Therapeutics,
Charlottesville, VA; 2 Virginia Commonwealth University,
Richmond, VA
NASH has emerged as the most common cause of chronic liver
disease in the Western world. Recently, obeticholic acid (OCA)
a semi-synthetic bile acid derivative and farnesoid-X-receptor
(FXR) agonist has been shown to improve insulin sensitivity in
humans with type 2 diabetes and liver histology in subjects
with NASH. The precise mechanisms by which these effects
are mediated are not fully known. AIMS: To use an unbiased
transcriptomic analysis of hepatocytes exposed to clinically relevant
doses of OCA to evaluate its effects on pathophysiologically
relevant pathways. METHODS: We previously described
a dynamic 3-dimensional system using liver-derived blood flow
and transport parameters to restore primary human hepatocyte
biology, allowing for culture at close to physiological insulin/
glucose conditions and eliciting drug responses at clinically-relevant
concentrations. This system represents an advance over
existing in vitro systems, which require supra-physiological insulin
concentrations and are associated with loss of polarity and
transport functions. We applied this system to gain insights into
the pharmacotoxicological effects of OCA. Primary hepatocytes
from 5 human donors were exposed to OCA for 48 hours
at concentrations approximating clinical therapeutic (0.5 μM)
and supratherapeutic (10 μM) levels. Whole genome transcriptomics
was performed by RNAseq, and the data analyzed
using both unbiased algorithms and biased interrogation of
specific pathways. RESULTS: A dose-dependent suppression
of Cyp7a and Cyp27a along with upregulation of bile salt
canalicular export transporters ABCB4 and ABCB11 and basolateral
transporters OSTA and OSTB were noted as expected.
Interestingly, FGF19, not known to be highly expressed in
hepatocytes, was strongly upregulated in our system by OCA
suggesting an autocrine suppression of FXR expression. A concomitant
suppression of LXR and HNF4α signaling associated
with NROB2 activation was also seen. Pleiotropic effects of
OCA included suppression of TGFβ as well as TNFα signaling
pathways, and could explain its beneficial effect of reducing
inflammatory and fibrotic changes in NASH. Consistent with
the hypercholesterolemia reported with OCA treatment was the
upregulation of ApoB, the major lipoprotein of LDL. CONCLU-
SIONS: OCA, in clinically relevant conditions, induces metabolic,
anti-inflammatory and anti-fibrotic/oncogenic signaling
in primary hepatocytes. These findings provide a mechanistic
basis for OCA effects in NASH.
Disclosures:
Arun J. Sanyal - Advisory Committees or Review Panels: Bristol Myers, Gilead,
Genfit, Abbott, Ikaria, Exhalenz; Consulting: Salix, Immuron, Exhalenz, Nimbus,
Genentech, Echosens, Takeda, Merck, Enanta, Zafgen, JD Pharma, Islet
Sciences; Grant/Research Support: Salix, Genentech, Intercept, Ikaria, Takeda,
GalMed, Novartis, Gilead, Tobira; Independent Contractor: UpToDate, Elsevier
Robert Figler - Employment: HemoShear Therapeutics, LLC
Brett R. Blackman - Board Membership: HemoShear LLC; Management Position:
HemoShear LLC; Patent Held/Filed: HemoShear LLC; Stock Shareholder:
HemoShear LLC
Svetlana Marukian - Employment: HemoShear LLC
Mark Lawson - Employment: Hemoshear
Aaron J. Mackey - Employment: HemoShear, LLC
David Manka - Employment: HemoShear Therapeutics; Stock Shareholder:
HemoShear Therapeutics
Brian R. Wamhoff - Stock Shareholder: HemoShear, LLC
Ajit Dash - Employment: HemoShear LLC
The following authors have nothing to disclose: Sol Collado
176
Inhibition of NF-kB by deoxycholic acid induces miR-
21/PDCD4-dependent hepatocelular apoptosis
Pedro M. Rodrigues 2 , Marta B. Afonso 2 , André L. Simão 2 , Pedro
M. Borralho 1 , Cecília M. Rodrigues 1 , Rui E. Castro 1 ; 1 iMed.
ULisboa & Dep. of Biochemistry and Human Biology, Faculty of
Pharmacy, University of Lisbon, Lisboa, Portugal; 2 iMed.ULisboa,
Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
MicroRNAs (miRNAs/miRs) play a key regulatory role in
metabolic liver function. In particular, miR-21 deregulated
expression has been associated with a wide spectrum of liver
disorders, contributing to disease development and progression.
We have demonstrated that deoxycholic acid (DCA), a
cytotoxic bile acid implicated in the pathogenesis of non-alcoholic
fatty liver disease, inhibits miR-21 expression in hepatocytes.
Still, the regulatory mechanisms behind miR-21 inhibition
and its contribution for cell demise are lacking. We aimed to
unveil the mechanisms underlying modulation of miR-21 by
DCA and to evaluate their exact contribution to DCA-induced
cell death. Primary rat hepatocytes were treated with 25-200
mM DCA for 24 h. Cell death, viability and caspase-3 activity
were measured by the ApoTox-Glo Triplex Assay. miR-21
expression was measured by qRT-PCR. Programmed cell death
4 (PDCD4), a miR-21 pro-apoptotic target, was evaluated by
immunoblotting and after transfecting cells with a reporter luciferase
plasmid. NF-kB, IkB and active caspase-2 levels were
also measured by immunoblotting, while NF-kB activation was
evaluated using a specific luciferase assay and by analyzing
NF-kB subcellular localization. Reactive oxygen species (ROS)
levels were analysed using the fluorescent probe 2’,7’-dichlorodihydrofluorescein
diacetate. Finally, for functional studies,
miR-21, PDCD4, caspase-2 and NF-kB were modulated using
both genetic and pharmacologic approaches, and a well-established
antioxidant, N-acetyl-L-cysteine (NAC). Our results
show that DCA inhibits miR-21 expression in a dose-dependent
manner, while increasing PDCD4 protein levels, with a
concomitant decrease in cell viability and an increase in cell
death, caspase-2/-3 activation and ROS production. Either
miR-21 overexpression or PDCD4 silencing hampered DCA-induced
cell death. Furthermore, NF-kB activity was decreased in
a similar pattern to miR-21 expression in DCA-treated hepatocytes.
In fact, NF-kB inhibition, using a selective chemical inhibitor
(BAY 11-7085), further decreased miR-21 and increased
PDCD4 expression levels and apoptosis by DCA. In agreement,
opposite results were observed in cells overexpressing
NF-kB or incubated with NAC. In conclusion, NF-kB represents
a major target of DCA in regulating the miR-21/PDCD4 pathway
whereas, in turn, oxidative stress and caspase-2 activation
are two key upstream mechanisms leading to inhibition of
NF-kB transcriptional activity by DCA. As such, these signaling
circuits constitute appealing targets for bile acid- and/or
miR-21-associated liver pathologies.
Disclosures:
The following authors have nothing to disclose: Pedro M. Rodrigues, Marta B.
Afonso, André L. Simão, Pedro M. Borralho, Cecília M. Rodrigues, Rui E. Castro