studies

900A AASLD ABSTRACTS HEPATOLOGY, October, 2015
1413
Versican: A Novel Modulator of Hepatic Fibrosis
Terence N. Bukong 1 , Sean B. Maurice 1,2 , Barinder Chahal 2,1 ,
David Schaeffer 2 , Paul J. Winwood 2,1 ; 1 Northern Medical Program,
University of Northern BC, Prince George, BC, Canada;
2 University of British Columbia, Vancouver, BC, Canada
Little is known about the deposition and turnover of proteoglycans
in liver fibrosis, despite their abundance in the extracellular
matrix. Versican plays diverse roles in modulating cell
behaviour in other fibroproliferative diseases, but remains
poorly described in the liver. Hepatic fibrosis was induced by
carbon tetrachloride (CCl 4
) treatment of C57BL/6 mice over
4 weeks followed by recovery over a 28 day period. Primary
mouse hepatic stellate cells (HSCs) were activated in culture
and versican was transiently knocked down in human (LX2)
and mouse HSCs. Expression of versican, A Disintegrin-like and
Metalloproteinase with Thrombospondin-1 motifs (ADAMTS)-1,
-4, -5, -8, -9, -15 and -20, and markers of fibrogenesis were
studied using qRT-PCR and Western blotting. CCl 4
treatment
led to significant increases in versican expression and the proteoglycanases
ADAMTS-5, -9, -15 and -20, alongside TNF-α,
alpha-smooth muscle actin (α-SMA), collagen-1, and TGF-β
expression. During recovery, expression of many of these genes
returned to control levels. However, expression of ADAMTS-5,
-8, -9 and -15 showed delayed increases in expression at 28
days of recovery which corresponded with decreases in versican
V0 and V1 cleavage products (G1-DPEAAE 1401 and
G1-DPEAAE 441 ). Activation of primary HSCs in-vitro significantly
increased versican, α-SMA, and collagen-1 expression.
Transient knockdown of versican in HSCs led to decreases in
markers of fibrogenesis and reduced cell proliferation, without
inducing apoptosis. Versican expression increases during
HSC activation and liver fibrosis, and proteolytic processing
occurs during the resolution of fibrosis. Knockdown studies in
vitro suggest a possible role of versican in modulating hepatic
fibrogenesis.
Disclosures:
The following authors have nothing to disclose: Terence N. Bukong, Sean B.
Maurice, Barinder Chahal, David Schaeffer, Paul J. Winwood
1414
Hepatic stellate cell-derived retinoid enhances functional
maturity of human embryonic stem cell-derived hepatocytes
on drug metabolism
Hyuk-Soo Eun, Wonhyo Seo, Won-IL Jeong; KAIST, Daejeon,
Korea (the Republic of)
Background: Human embryonic stem cell-derived hepatocyte
(hES-Hep) could be used for the screening tool of drug hepatotoxicity.
However, drug metabolic functions of hES-Hep
are still poor. Retinoids including retinol and its metabolites
play important roles not only in organ development but also
metabolic regulation and they are enriched in hepatic stellate
cells (HSCs) of liver. Therefore, in this study, we evaluate the
effects of mouse HSCs (mHSCs) on the expression and activity
of drug metabolic enzymes in hES-Hep. Methods: Analyses
of microarray, co-culturing with mHSCs or human HSC cell
lines (LX-2 and hTERT), retinoid treatment, flow cytometry analysis,
protein and gene expression, and drug metabolic activity
were performed in hES-Hep. Results: In microarray analyses,
gene expression of drug metabolic enzymes such as CYP1A2,
CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in hES-Hep was
specifically down-regulated compared with normal hepatocyte.
However, that expression was significantly increased in
co-cultured hES-Hep with retinol-storing mHSCs compared to
control hES-Hep. In co-cultured mHSCs, expression of LRAT was
down-regulated while expression of retinaldehyde dehydrogenase-1
(Raldh1) and cellular retinoic acid binding protein-1
was significantly increased, indicating decreased retinol storage
but increased production and delivery of retinoic acids in
HSCs. Similarly, direct treatments of retinoic acids increased
expression of metabolic enzymes in hES-Hep compared with
control hES-Hep. In contrast, retinoic acid deficiency in LX-2,
hTERT and Raldh1-depleted HSCs did not affect gene expression
of metabolic enzymes in hES-Hep, suggesting that retinoic
acid might be a critical factor for functional maturation of hES-
Hep. In flow cytometry, two biggest populations of hES-Hep
depending on cell size and granularity expressed major types
of drug metabolic enzymes and one smallest population still
highly expressed mesodermal markers such as SOX17, FOXA2
and GATA4. In western blotting, protein levels of CYP1A2,
CYP2E1, and CYP3A4 were increased in co-cultured hES-Hep
with retinol-storing HSCs. Consistently, metabolic activities of
CYP1A2 (phenacetin), CYP3A4 (testosterone) and CYP2E1
(chlorozoxazone) were significantly improved in co-cultured or
retinoic acid-treated hES-Hep compared with controls. Conclusions:
Based on our data, retinol-storing mHSCs could enhance
functional maturation of hES-Hep through retinoic acid-mediated
increased expression and activity of drug metabolic
enzymes. Thus, co-culturing with mHSCs or treatments of retinoic
acids could be useful ways to adapt immature hES-Hep for
the application of drug toxicity screening system.
Disclosures:
The following authors have nothing to disclose: Hyuk-Soo Eun, Wonhyo Seo,
Won-IL Jeong
1415
Calcium Mobilization through L-type Channels in
Hepatic Stellate Cell is Essential for TGF-b-mediated
CTGF Induction in Pyk2-dependent Manner
Jonghwa Kim 1 , SoHee Kang 1 , Soohyun Park 1 , Ju-Yeon Cho 1 , Won
Sohn 1 , David A. Brenner 2 , Yong-Han Paik 1 ; 1 Samsung medical
center, Seoul, Korea (the Republic of); 2 UC San Diego, La Jolla,
CA
Background: In hepatic fibrogenesis hepatic stellate cell (HSC)
is a major cell type responsible for producing a major profibrogenic
cytokine TGF-b, and connective tissue growth factor
(CTGF), a major fibrogenic mediator in several organs. The
multi-functional nature of TGF-b signaling in hepatic fibrogenesis
is still elusive. At the previous AASLD (2014) we reported
that Pyk2 is essential for TGF-b-mediated, Smad-independent
CTGF induction. Pyk2 is known to be calcium-sensitive, and
TGF-b was reported to increase intracellular calcium level.
Therefore, we investigated if TGF-b activates Pyk2 by increasing
intracellular calcium levels through L-type voltage-gated
calcium channel in hepatic stellate cell. Methods: Immortalized
human stellate cell line, LX-2, has been cultured. After TGF-b
treatment, expression of CTGF and a-SMA were assessed with
RT-PCR and western blot. Pharmacological inhibitor and siR-
NA-mediated knockdown were used to modulate the activities
and expression levels of protein. Intracelllular calcium mobilization
was measured with Fura-2/AM. Activation of Pyk2
was addressed in western blot using different phosphorylation
site-specific antibodies. Results: CTGF expression was up-regulated
within 1hr in TGF-b stimulated LX-2. This up-regulation
was greatly suppressed by siRNA-mediated knockdown and
pharmacological inhibitor of Pyk2. TGF-b treatment increased
phosphorylation of Pyk2 on tyrosine 402, 579/580, and 881.
Consistent with the previous reports, TGF-b increased intracellular
calcium concentration in Fura-2-preloaded LX-2. CTGF