studies

376A AASLD ABSTRACTS HEPATOLOGY, October, 2015
325
CD45+ fraction in murine adipose tissue-derived stromal
cells is therapeutical to Concanavalin A-induced
murine acute hepatitis model.
Akihiro Seki 1 , Yoshio Sakai 2 , Masatoshi Yamato 1,2 , Kosuke
Ishida 1 , Hisashi Takabatake 1 , Alessandro Nasti 1 , Masao Honda 2 ,
Shuichi Kaneko 1,2 ; 1 Disease control and homeostasis, Kanazawa
university, Kanazawa, Japan; 2 Department of Gastroenterology,
Kanazawa university hospital, Kanazawa, Japan
Adipose tissue-derived stromal cells (ADSCs) are expected to
be useful in regeneration therapy for liver disease because of
their pluripotency and immunomodulatory capability. However,
ADSCs are composed of heterogeneous cell fractions, such as
mesenchymal stem cells, and the regenerative or therapeutic
effects of all ADSC cell fractions have not been fully elucidated,
particularly for liver diseases. In this study, we assessed
whether the substantial ADSC CD45+ leukocyte-lineage fraction
ameliorates the hepatic inflammatory disease condition
induced by injecting mice with Concanavalin A (ConA). [Materials
and Methods] ADSCs were obtained from inguinal subcutaneous
adipose tissue of C57Bl/6 male mice by collagenase
digestion. C57Bl/6 female mice were injected intravenously
with 200 mg ConA to generate the murine hepatitis model.
The CD45+ fraction, which was about 15% of all ADSCs, was
isolated with a cell sorter (FACSAriaTMII), and 5 × 10 4 CD45+
cells, 1 × 10 6 ADSCs, or PBS were injected intravenously into
C57BL/6 mice 3 hours after the ConA injection (n = 5 each).
Sera and liver tissues were obtained from the ConA-injected
mice after 16 hours to measure serum alanine aminotransferase
(ALT) and lactate dehydrogenase activities (LDH), and to conduct
immunohistochemical and gene expression analyses using
the real-time polymerase chain reaction. CD45+ cells (1 × 10 4
cells) were also co-cultured with 1 × 10 5 splenocytes stimulated
with ConA for 2 hours, followed by a gene expression analysis.
[Results] Injecting 2 × 10 4 CD45+ cells into the ConA-stimulated
hepatitis mice improved liver congestion compared with
that of the PBS control. Correspondingly, injecting CD45+ cells
into ConA-stimulated hepatitis mice suppressed serum ALT and
LDH activities (P = 0.02 each) but increased hepatic alpha
fetoprotein and albumin RNA expression (P = 0.01 and 0.02).
These therapeutic effects of the CD45+ cells injection on the
ConA-stimulated hepatitis mice were similar to those produced
after ADSCs were injected. The immunohistochemical analysis
showed that administrating CD45+ cells ameliorated the
numbers of CD4+, CD11b+, and Gr-1+ cells infiltrating the
livers of the ConA-stimulated hepatitis mice. Enhanced interferon-gamma,
tumor necrosis factor-alpha, and chemokine (C-C
motif) ligand 3 gene expression by ConA-stimulated splenocytes
was suppressed when they were co-cultured with CD45+
cells in vivo. Thus, the ADSC CD45+ fraction therapeutically
suppressed ConA-induced mouse hepatitis. [Conclusion] The
CD45+ leukocyte-lineage fraction in ADSCs, which is uniquely
immunosuppressive and beneficial, may have important therapeutic
effects in an acute hepatitis mouse model.
Disclosures:
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Akihiro Seki, Yoshio Sakai,
Masatoshi Yamato, Kosuke Ishida, Hisashi Takabatake, Alessandro Nasti,
Masao Honda
326
Cross-talk between autophagy and the transcription
factor KLF2 determines endothelial cell phenotype, liver
microvascular function and hepatic damage after acute
liver injury.
Sergi Guixé-Muntet 1 , Fernanda C. de Mesquita 1,3 , Sergi Vila 1 ,
Carmen Peralta 2 , Juan Carlos Garcia-Pagan 1 , Jaime Bosch 1 , Jordi
Gracia-Sancho 1 ; 1 Barcelona Hepatic Hemodynamic Lab, Hospital
Clinic de Barcelona - IDIBAPS - CIBEREHD, Barcelona, Spain;
2 IDIBAPS - CIBEREHD, Barcelona, Spain; 3 Laboratório de Biofísica
Celular e Inflamação, PUCRS, Porto Alegre-RS, Brazil
Background & aims: The transcription factor Kruppel-like Factor
2 (KLF2) can be induced by different drugs, including simvastatin,
and its expression confers a vasoprotective phenotype to
the endothelium. Considering recent data suggesting activation
of autophagy by statins, we aimed at: 1) characterizing the
molecular relationship between autophagy and KLF2 in the
endothelium, 2) assessing this relationship in acute liver injury
due to cold ischemia/warm reperfusion (I/R), and 3) studying
the effects of modulating KLF2-autophagy in in vitro and ex
vivo models of acute liver injury. Methods: 1) In vitro: Autophagy
and KLF2 were modulated and characterized in vascular
endothelial (HUVEC) and sinusoidal endothelial cells (LSEC)
cultured in standard conditions or under I/R. KLF2 was induced
by simvastatin and resveratrol or genetically regulated with
siRNA or adenovirus in the presence or absence of autophagy
modulators. Cell viability was evaluated by double-staining
with acridine orange and propidium iodide, and by trypan
blue exclusion. Autophagic flux was assessed by western blot
of LC3B, immunofluorescence of accumulation of autophagosomes,
and colocalization of autophagosomes & lysosomes. 2)
Ex vivo: Wistar rats received a) vehicle, b) simvastatin (1mg/
kg, i.v.) or c) chloroquine (inhibitor of autophagy; 60mg/kg,
i.p.) + simvastatin. Livers were explanted, cold stored in Wisconsin
solution (16h) and warm-reperfused (2h). Microvascular
function was assessed by liver vasorelaxation to acetylcholine.
Results: A positive feed-back between autophagy and KLF2
was observed: KLF2-inducers simvastatin and resveratrol, but
not the autophagy activator Rapamycin, caused endothelial
KLF2 overexpression through an autophagy-Rac1-rab7-dependent
mechanism. In turn, KLF2 induction promoted further
activation of autophagy. Cold ischemia blunted autophagic
flux in LSEC. Upon reperfusion, LSEC cold stored in Celsior
solution showed proper reactivation of autophagy. However,
LSEC stored in Wisconsin failed to reactivate autophagy, most
probably due to impairment in autophagosome and lysosome
fusion. Simvastatin re-activated autophagy by up-regulating
rab7 (restoring autolysosome formation), which resulted in
increased KLF2 levels, improved cell viability (in vitro), and
ameliorated hepatic damage and microvascular function (ex
vivo). Conclusions: We herein describe for the first time the
complex link between autophagy and KLF2 modulating the
phenotype and survival of the endothelium. These results help
understanding the underlying molecular mechanisms of the
protection conferred by KLF2-inducers, such as simvastatin, in
hepatic and extra-hepatic vascular disorders.
Disclosures:
Juan Carlos Garcia-Pagan - Consulting: Novartis; Grant/Research Support:
GORE
Jaime Bosch - Consulting: Falk, Gilead Science, Intercept Therapeutics, Conatus
Pharmaceuticals, Exalenz, Almirall, Chiasma
The following authors have nothing to disclose: Sergi Guixé-Muntet, Fernanda C.
de Mesquita, Sergi Vila, Carmen Peralta, Jordi Gracia-Sancho