studies

306A AASLD ABSTRACTS HEPATOLOGY, October, 2015
192
Circulating exosomes from healthy mice attenuate
hepatic stellate cell activation and are anti-fibrotic in
vivo
Li Chen 1 , Ruju Chen 1 , Sherri Kemper 1 , David Brigstock 1,2 ; 1 Clinical
& Translational Research, Nationwide Childrens Hospital, Columbus,
OH; 2 Surgery, The Ohio State University, Columbus, OH
Exosomes are 50-150 nm membranous vesicles that shuttle a
complex molecular cargo (miRs, mRNAs, proteins) between
producer and recipient cells resulting in epigenetic regulation
of cell function. After their release from producer cells, exosomes
traverse intercellular spaces and may either be taken
up by neighboring cells or enter body fluids and be dispersed
systemically. The goal of our work was to determine if hepatic
stellate cells (HSC) are regulated by circulating exosomes.
Exosomes were purified from the serum of healthy or fibrotic
Swiss Webster male mice and tested for their effect on (i)
activation or fibrogenesis in primary cultures of mouse HSC,
or (ii) CCl 4
-induced liver injury in mice. Exosomes (10μg/ml)
from the serum of healthy mice resulted in decreased CTGF,
αSMA or collagen α1(I) mRNA after treatment of D9 HSC for
24hrs (p < 0.01) whereas transcript expression was increased
or unaffected when the cells were exposed to serum exosomes
from fibrotic mice. In a 10-day CCl 4
injury model in
male BAC reporter transgenic (TG) CTGF-EGFP FX156GSat/
Mmucd Swiss Webster mice expressing GFP under the control
of the CTGF promoter, hepatic GFP or αSMA expression was
attenuated by i.p. administration every other day (40μg/g) of
serum exosomes from healthy mice, but not from fibrotic mice
(p < 0.01). In 5-wk CCl 4
fibrosis models, i.p. administration of
serum exosomes (40 μg/g) from healthy mice during the last 2
wks of CCl 4
treatment caused a dramatic and dose-dependent
decrease in hepatic GFP-CTGF production. This was associated
with decreased expression of CTGF, αSMA or collagen
mRNA or protein (p < 0.01). The same effects occurred in
wild type mice. To identify the principal miRs in serum exosomes
that are differentially expressed between normal versus
fibrotic mice, miR profiling was performed on serum exosomes
using a miRnome miR PCR Array. Non-biased ranking of the
data identified miR-23b, -34c, -106a, -151, -200b, -455, -483
-532, -653, and -687 as the principal differentially expressed
miRs and as candidate anti-fibrotics with the ability to attenuate
expression of CTGF, αSMA or collagen in cultured HSC. These
studies show that circulating exosomes from healthy individuals
are instrinsically anti-fibrotic and that this property is attributable,
at least in part, to specific miR constituents. We propose
that circulating exosomes offer a novel platform for liver fibrosis
therapy and that continued interrogation of their complex
molecular payload will result in the identification of additional
therapeutic candidates.
Disclosures:
David Brigstock - Stock Shareholder: FibroGen
The following authors have nothing to disclose: Li Chen, Ruju Chen, Sherri Kemper
193
Combining high-throughput genome-wide RNAi and
computational pharmacological screening identifies
glyburide as a potent therapeutic candidate for liver
disease due to alpha-1-antitrypsin deficiency
Yan Wang, Murat C. Cobanoglu, Jie LI, Pamela Hale, Tunda Hidvegi,
Micheal Ewing, Andrew Chu, Gary A. Silverman, Stephen C.
Pak, Ivet Bahar, David H. Perlmutter; Departments of Pediatrics and
Computational Biology, University of Pittsburgh School of Medicine,
Pittsburgh, PA
Alpha-1-antitrypsin deficiency (ATD) causes severe liver disease
by the pathologic accumulation of the misfolded alpha-1-antitrypsin
Z (ATZ) in hepatocytes. Liver transplantation is the only
therapy currently available for this liver disease. In this study
we investigated the possibility of discovering novel therapeutic
drug candidates by interrogating a C. elegans model of ATD
with genome-wide RNAi screening and computational (druggene
interaction) pharmacological strategies. High-content
genome-wide RNAi screening of our C. elegans model of ATD
was utilized to identify genes that could modify the proteotoxicity
of ATZ. Out of more than 16,000 RNAi, the initial screen
identified 104 genes that are potential positive modifiers of
ATZ proteotoxicity. These genes were then analyzed as potential
targets of known drugs using the DrugBank 3.0 computational
database. One of the drugs identified by this approach
is glyburide (glibenclamide, GLB), a sulfonylurea drug that has
been used broadly in clinical medicine as an oral hypoglycemic
agent. In this study, we show that GLB mediates a marked
decrease in steady-state levels of misfolded ATZ in a mammalian
cell line model of ATD and that this effect is dose- and
time-dependent with an optimal dose of 40 mM. Pulse-chase
labeling experiments indicate that GLB specifically and selectively
mediates an increase in the rate of intracellular degradation
of ATZ. Mechanistic studies suggest that GLB increases
autophagic flux but does not affect the phosphorylation of
mTOR substrates and so its effect on the autophagy pathway is
TOR-independent. As GLB is known to induce cellular calcium
influx via action on the ATP-sensitive potassium channel, we
investigated whether GLB could affect the intracellular accumulation
of ATZ by acting on intracellular calcium signaling
and found that calcium chelator BAPTA-AM partially blocks the
GLB-mediated reduction of ATZ levels. Most importantly, GLB
reduces hepatic ATZ load and hepatic fibrosis when administered
systemically to the PiZ mouse model of ATD. Preliminary
studies of GLB analogues suggest that its effects on degradation
of ATZ can be dissociated from its effects on insulin secretion.
In conclusion, these results indicate: 1) GLB reduces the hepatic
proteotoxicity of ATZ accumulation by a calcium-dependent,
mTOR-independent effect on intracellular degradation; 2) GLB
(analogues) is a very appealing therapeutic drug candidate for
ATD because of its wide margin of safety; 3) interrogation of
a genetically tractable disease model by combining computational
with high-content automated RNAi screening strategies is
a valid and powerful drug discovery platform.
Disclosures:
The following authors have nothing to disclose: Yan Wang, Murat C. Cobanoglu,
Jie LI, Pamela Hale, Tunda Hidvegi, Micheal Ewing, Andrew Chu, Gary A.
Silverman, Stephen C. Pak, Ivet Bahar, David H. Perlmutter