studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 465A
511
Cell-Type-Specific Expression Profile of Repopulating
Hepatocytes by Translating Ribosome Affinity Purification
(TRAP)
Kirk J. Wangensteen 1,2 , Amber W. Wang 2 , Monica Teta-Bissett 2 ,
Klaus H. Kaestner 2 ; 1 Gastroenterology, University of Pennsylvania,
Philadelphia, PA; 2 Genetics, University of Pennsylvania, Philadelphia,
PA
Understanding the genetic regulation of liver regeneration
could help in the development of new treatments for liver disease.
Much of the research on liver regeneration has focused
on the partial hepatectomy model in rodents; however, the lack
of cell necrosis and immune response calls for the development
of novel paradigms that recapitulate human liver diseases. Fah -
/-
mice, a model of hereditary tyrosinemia, are suitable for the
study of liver repopulation after injury because repopulating
hepatocytes can be genetically traced. We first injected Fah -
/-
mice with plasmids that co-express Fah and GFP, and found
that we could isolate GFP-positive hepatocytes by fluorescence
activated cell sorting, but met challenges in isolating pure populations
of repopulating hepatocytes in the setting of injury. We
then employed a different approach, the Translating Ribosome
Affinity Purification (TRAP) system, to isolate mRNA specifically
from repopulating hepatocytes. We injected Fah -/- mice with
a plasmid that co-expresses Fah and a fusion of GFP and the
ribosomal subunit L10a, and then induced liver injury for 1 or
4 weeks. Liver tissue harvested from these mice was immediately
lysed and polysomes were extracted. Anti-GFP antibodies
were then used to affinity-purify the mRNAs bound to the GFP-
L10a fusion protein expressed within repopulating hepatocytes
(Figure). No RNA was obtained from wildtype control liver,
as expected. We performed high throughput mRNA sequencing
(RNA-seq) to identify differentially expressed genes during
repopulation as compared to quiescent hepatocytes. Gene
ontology analysis showed that the genes were enriched in metabolic
pathways and in c-Myc-responsive genes. In conclusion,
this represents the first analysis of the differential expression
of repopulating hepatocytes in an injury model. Intriguingly,
liver repopulation involves differential regulation of metabolic
genes, likely to maintain metabolic homeostasis in the setting
of widespread liver injury.
Bioanalyzer tracings of affinity-purified RNA from mice treated
with (Green and Red), or without (Blue) the TRAP vector, illustrating
the specificity of the system.
512
In vivo distribution of exosome packaged extracellular
miRNA-155 to the liver, hepatocytes and Kupffer cells
and its functional effects
Shashi Bala 1 , Timea Csak 1 , Fatemah Momen-Heravi 1 , Dora Lippai
1 , Karen Kodys 1 , Donna Catalano 1 , Abhishek Satishchandran 1 ,
Victor Ambros 2 , Gyongyi Szabo 1 ; 1 Medicine, UMass Medical
School, Worcester, MA; 2 Molecular Medicine, UMass Medical
School, Worcester, MA
Purpose: Circulating miRNAs are gaining increasing interest
as new biomarkers of diseases and means of intercellular communication.
Previously, we showed induction of miR-155 in the
plasma and found that miR-155 was associated with extracellular
vesicles (EVs). However, little is known about the half-life
and biodistribution of EVs associated miRNAs in vivo. Here, we
established an exosome-based miR-155 mimic delivery system
to study the biodistribution and half-life of EV associated miR-
155 using a miR-155 knock out (KO) mouse model. Methods:
Female wild type (WT) (C57/BL6J) or miR-155 KO mice were
used. Exosomes were isolated from murine B cells using filtration
method and characterized by electron microscopy, Nanosight
and Western blot analyses. Electroporation was used
to introduce miR-155 mimic into exosomes. Purified miR-155
or control mimic loaded exosomes were administered intravenously
to miR-155 KO mice. Mice were perfused to eliminate
blood contamination. The Mann-Whitney test was employed
for statistical analysis. Results: Our results indicate that administration
of exosomes loaded with miR-155 mimic resulted in a
rapid accumulation of miR-155 in the plasma of recipient miR-
155 KO mice with subsequent distribution in the liver, adipose
tissue, lung, muscle and kidney (highest to lowest, respectively).
At the cellular level, miR-155 was detected in hepatocytes and
liver mononuclear cells of recipient miR-155 KO mice in vivo,
suggesting successful uptake of exosomal miR-155 by various
cell populations of the liver. Functionally, exosomes-mediated
restoration of miR-155 in Kupffer cells isolated from miR-155
KO mice resulted in the induction of pro-inflammatory cytokines
(MIP2 and MCP1) after LPS challenge in an in vitro co-culture
system. To mimic the half-life and distribution of miRNA naturally
present in the plasma, we administered (iv) WT plasma
(donor) to miR-155 KO (recipient) mice. WT plasma was generated
from CpG+LPS treated WT mice and was enriched in miR-
155. Our results indicate detection of donor miR-155 in plasma
of recipient miR-155 KO mice and distribution to the liver and
adipose tissue. Induction of pro-inflammatory cytokines was
found in recipient miR-155 KO mice suggesting some biological
activity. In summary, our results demonstrate tissue biodistribution
and biologic function of EV-associated miR-155.
Conclusion: Exosome packaged miR-155 is readily distributed
to the liver in a cell–specific manner resulting in KC activation.
These observations will aid in developing new exosome-mediated
miRNA delivery and investigating biological function of
other extracellular miRNAs in vivo.
Disclosures:
The following authors have nothing to disclose: Shashi Bala, Timea Csak,
Fatemah Momen-Heravi, Dora Lippai, Karen Kodys, Donna Catalano, Abhishek
Satishchandran, Victor Ambros, Gyongyi Szabo
Disclosures:
The following authors have nothing to disclose: Kirk J. Wangensteen, Amber W.
Wang, Monica Teta-Bissett, Klaus H. Kaestner