studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 887A
ulator of HSCs activation in liver fibrogenesis. Nowadays,
the epigenetic machinery embraces an extensive network of
non-coding RNAs (ncRNAs), such as long non coding RNAs
(lncRNAs) and microRNAs (miRNAs). The transcription of
ncRNAs establishes new regulatory systems affecting chromatin
structure and gene expression. The aim of this study was to
identify potential targets and a novel ncRNA regulatory network
controlled by MeCP2 in HSCs. Materials and methods:
RNA from Wt (n=3) and MeCP2-null (n=3) mice HSCs was
subjected to a microarray based profiling analyses (Arraystar
Mouse LncRNA Microarray V2.0 platform) and to a small RNA
next-generation sequencing (NGS) (Illumina platform). Results:
Using the microarray platform we found 161 upregulated and
83 downregulated lncRNAs in MeCP2-null HSCs. We focused
our attention to Gm3893 and AK080187; their deregulated
expression was confirmed by qPCR and correlated with the
expression of their associated gene transcripts (cytokines Ccl-
21b/c and IL11ra2 receptor and the profibrogenic marker
Acta2, respectively). Pathway analysis based on KEGGs
database denoted that most of 284 downregulated mRNAs
in MeCP2-null HSCs were significantly enriched at DNA replication
and cell cycle pathways, while most of 124 upregulated
mRNAs were associated with pathways involved in the
immune system and metabolism. Most importantly, MeCP2-
null HSCs presented a robust downregulation in five members
of the MCM protein complex and a remarkable decrease in
other components of the replisome multisubunit complex. The
resulting altered pattern of mRNAs also emphasized the downregulation
of the hyaluronan synthase 2 (Has2) upon loss of
MeCP2 in HSCs. In liver, Has2 was predominantly expressed
in RNA of HSCs and its protein expression increased over
the HSCs transdifferentiation process. In primary HSCs, small
interfering RNA for Has2 decreased the gene expression of
ECM proteins. Of note, miRNA-328-3p pinpointed from the
14 significantly altered miRNA identified by NGS analyses
as has been proved to target and reduce the hyaluronic acid
receptor CD44 expression. Conclusions: Our findings successfully
evidence a vast ncRNA network controlled by MeCP2
and highlight the complex role that MeCP2 elicits in the HSC
transdifferentiating process.
Disclosures:
The following authors have nothing to disclose: EVA MORAN-SALVADOR, Pier P.
Paoli, Graham R. Smith, Agata Page, Rachel M. Howarth, Fiona Oakley, Jelena
Mann, Derek Mann
1382
Cartilage oligomeric matrix protein participates in the
pathogenesis of liver fibrosis
Fernando Magdaleno 1,2 , Elena Arriazu 2 , Marina Ruiz de Galarreta
2 , Yu Chen 1 , Xiadong Ge 1,2 , Laura Conde de la Rosa 2 , Natalia
Nieto 1,2 ; 1 Pathology, University of Illinois at Chicago, Chicago,
IL; 2 Medicine, Icahn School of Medicine at Mount Sinai, New
York, NY
BACKGROUND & HYPOTHESIS: Liver fibrosis is characterized
by excessive accumulation of extracellular matrix (ECM) proteins,
mainly fibrillar collagen-I, as a result of persistent liver
injury. Cartilage oligomeric matrix protein (COMP) is a glycoprotein
typically found in the ECM of skeletal tissue. Increased
COMP expression has been associated with fibrogenesis in
systemic sclerosis, lung fibrosis and chronic pancreatitis. Since
COMP is expressed in cirrhotic livers and it is significantly
induced in hepatocellular carcinoma we hypothesized that
COMP could induce fibrillar collagen-I protein deposition and
participate in matrix remodeling contributing to the pathophysiology
of liver fibrosis. METHODS: Chronic thioacetamide (TAA)
administration (4 mos) and carbon tetrachloride (CCl 4
) injection
(1 mo) were used as models to induce liver fibrosis in
wild-type (WT) littermates and in global Comp -/- mice. Controls
received water or mineral oil, respectively. To dissect the mechanism
for COMP signaling, in vitro experiments were carried
out with primary rat hepatic stellate cells (HSCs) and qPCR,
gelatin zymography or western blot analysis were performed.
To determine if physical interaction occurred between COMP
and collagen-I and how this conditioned collagen-I cleavage
by matrix metalloproteinases (MMPs), in vitro reconstituted systems
were used followed by immunoprecipitation and immunoblotting.
RESULTS: COMP expression was detected in livers
from control WT littermates and was up-regulated in response
to chronic TAA- or CCl 4
-induced liver injury. TAA-treated or
CCl 4
-injected Comp -/- mice showed less liver damage (ALT and
AST activities, necrosis, inflammation and hepatocyte ballooning
degeneration) and fibrosis compared to their matching
control WT littermates. Challenge of HSCs with recombinant
COMP (rCOMP) up-regulated intra- and extracellular collagen-I
protein expression and increased MMP2, 9 and 13. However,
rCOMP neither affected TGFβ protein, a well-known pro-fibrogenic
factor, nor altered Tnfα nor tissue inhibitor of metalloproteinase-1
(Timp1) mRNAs. Moreover, COMP showed major
ability to bind collagen-I; yet, the binding did not preclude from
cleavage by MMP1, the MMP with the highest affinity for collagen-I
cleavage. Last, we identified that rCOMP significantly
up-regulated collagen-I protein expression in HSCs via CD36
receptor binding and activation of the MEK1/2-pERK1/2
signaling pathway. CONCLUSION: These results suggest that
COMP contributes to liver fibrosis by up-regulating collagen-I
protein synthesis but not its cleavage by MMP1; thus, resulting
in significant collagen-I deposition.
Disclosures:
The following authors have nothing to disclose: Fernando Magdaleno, Elena
Arriazu, Marina Ruiz de Galarreta, Yu Chen, Xiadong Ge, Laura Conde de la
Rosa, Natalia Nieto
1383
IL-10-producing regulatory B cells from chronic hepatitis
B patients suppressed CD4+T cells proliferation and
enhanced regulatory T cells function
Li-sha Cheng, Yun Liu, Wei Jiang; Gastroenterology, Zhongshan
hospital, Shanghai, China
Background & aims: Chronic hepatitis B (CHB) infection is a
leading cause of liver fibrosis, cirrhosis and even hepatocellular
carcinoma. Although hepatitis B virus (HBV) itself is non-cytopathic,
both HBV-related liver damage and viral control are
immune-mediated. There were abundant evidences to believe
that regulatory T (Treg) cells play pivotal roles in the immunopathogenesis
of HBV-related liver diseases. Nowadays, a
new subset of IL-10-producing B cells has been shown to play
vital roles in modulating autoimmune diseases. Here, we evaluated
the role of regulatory B (Breg) cells in the pathogenesis
of HBV-related liver fibrosis (HBV-LF) and assess their impact
on the proliferation of CD4+T cells the immunoregulatory
effects on Treg cells. Methods: A total of 67 treatment-naïve
patients who were diagnosed as chronic HBV (CHB) and
25 healthy donors took part in the study. Firstly, we determined
the changes in number and function of Breg cells in
CHB patients compared with that of healthy controls. For the
in-vitro experiments, purified CD4+T cells were cultured alone
or with autologous Breg cells and their proliferation response
was determined by the thymidine method. Simultaneously, to
elucidate the exact effects of Bregs on Tregs and the potential
methanism, human Breg and Treg cells were co-cultured in the