studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 847A
1292
Implants of matrix-embedded endothelial cells rescue
ischemic tissue and liver engraftment in hepatectomized
mice
Pedro Melgar-Lesmes 1 , Mercedes Balcells 2,1 , Elazer R. Edelman 1,3 ;
1 Edelman Lab, Institute for Medical Engineering and Science,
Massachusetts Institute of Technology, Cambridge, MA, USA,
Cambridge,, MA; 2 Bioengineering department, Institut Químic de
Sarrià, Ramon Llull Univ, Barcelona, Spain; 3 Cardiovascular Division,
Brigham and Women’s Hospital, Harvard Medical School,
Boston, MA
Background and aims: Ischemia during liver transplantation
promotes a cascade of cellular injury responses that lead to
inflammation, cell death, and organ dysfunction. Matrix-embedded
endothelial cells (MEECs) have immunomodulatory
effects in vitro and in vivo. Therefore we investigated the benefits
of implants of MEECs as modulators of inflammation and
regeneration after liver engraftment in mice. Methods: Partial
hepatectomy (70%) was performed by excising the left lobe
and half the median lobe of the mouse liver. Four groups of
10 animals were distributed as follows: 1) acellular matrices
in the interface between the remaining median lobe and an
autograft from the left lobe; 2) MEECs between the remaining
median lobe and an autograft from the left lobe; 3) acellular
matrices between the remaining median lobe and an allograft
from the left lobe of another mouse; 4) MEECs between the
remaining median lobe and an allograft from the left lobe of
another mouse. Vascular architecture was analysed using angiography
with FITC dextran 2000 kDa by fluorescent multiphoton
microscopy 7 days post-op. Hepatic DNA fragmentation
and apoptosis were quantified in the median lobe and grafts
by TUNEL assay and Western Blot of activated caspase 3,
respectively. Serum markers of liver damage Alanine Aminotransferase
(ALT) and Aspartate Aminotransferase (AST) were
quantified in all animal groups. The phenotype of lymphocyte
subsets in the liver after implantation of allografts was quantified
by gene expression of Th1 (interferon gamma: INFγ; interleukin
2: IL-2) and Th2 (interleukin 4: IL-4; interleukin 10: IL-10)
genes by Real-Time PCR. Results: Implants with MEECs created
a functional vascular splice between the ischemic median lobe
and autografts or allografts improving the rate of liver donor
regeneration by 15% compared to acellular controls. MEECs
prevented apoptosis by 85% in donor liver lobes, by 85%
in autologous grafts and by 79% in allogeneic engraftments.
As a result, serum levels of ALT and AST were significantly
reduced after autologous or allogeneic engraftments. The beneficial
immunomodulatory effects of MEECs promoted allograft
immunotolerance expressed as a significant reduction of Th1
(INFγ and IL-2) and increase of Th2 (IL-4 and IL-10) cytokine
expression. Conclusions: Implants of MEECs are endothelial
cell-based scaffolds with potential to be used to improve current
surgical procedures in liver transplantation and to reduce
ischemic and inflammatory disorders. Their application may
contribute obtaining functional liver grafts of adequate size for
the recipient without subjecting the donor to undue risk.
Disclosures:
The following authors have nothing to disclose: Pedro Melgar-Lesmes, Mercedes
Balcells, Elazer R. Edelman
1293
Identification of liver monocytic myeloid-derived suppressor
cells and elucidation of their roles in non-alcoholic
fatty liver disease
Liying Yao, Masanori Abe, Teruki Miyake, Yoshiko Nakamura,
Yusuke Imai, Yohei Koizumi, Takao Watanabe, Osamu Yoshida,
Masashi Hirooka, Yoshio Tokumoto, Bunzo Matsuura, Yoichi
Hiasa; Department of Gastroenterology and Metabology, Ehime
Universiy Graduate School of Medicine, Ehime, Japan
Background/Aim: Myeloid-derived suppressor cells (MDSCs)
comprise a heterogeneous population of myeloid cells and are
recognized as suppressors of T cell functions. In mice, these
cells identified by the co-expression of CD11b and Gr-1 surface
markers. Previous studies revealed that liver MDSC exhibit
phenotypic and functional diversity; however, information
regarding their characteristics and mechanisms of suppression,
reported in these studies, was conflicting in nature. Immune
responses may contribute to the pathogenesis of non-alcoholic
fatty liver disease (NAFLD); however, little is known regarding
the role of myeloid regulatory cells. In this study, we characterized
the phenotype of liver MDSCs in a murine model of
NAFLD and explored the mechanism underlying their immunosuppression.
Methods: C57BL/6 mice were fed a normal
diet (ND) or a high-fat diet (HFD) for 12 months. Different subtypes
of CD11b + Gr-1 + cells were sorted from liver non-parenchymal
cells by FACS. CD11b + Gr-1 + cells were co-cultured
with T cells in the presence of anti-CD3 beads or allogeneic
dendritic cells, and suppressive functions were investigated
using a carboxyfluorescein succinimidyl ester assay. Nitric
oxide (NO) concentrations in culture supernatants were measured
using Griess reagent. In some experiments, L-NIL, an
inducible NO synthase (iNOS) inhibitor, was added at the
start of the culture. Results: In the livers of HFD-fed mice, the
frequency of CD11b + Gr1 dim cells (monocytic myeloid cells),
but not CD11b + Gr1 hi cells, was higher than that in the livers
of ND-fed mice over time. CD11b + Gr1 dim cells were divided
into SSC high and SSC low populations. The frequency of SSC low-
CD11b + Gr1 dim cells increased in the livers of HFD-fed mice
to a greater extent than that observed in the livers of ND-fed
mice. In addition, SSC low CD11b + Gr1 dim cells suppressed T cell
proliferation in a dose-dependent manner; however, this suppression
was not observed in SSC low CD11b + Gr1 high cells. We
also found that SSC low CD11b + Gr1 dim cells expressed iNOS
after co-culture with T cells, and the supernatants obtained from
the co-culture of SSC low CD11b + Gr1 dim cells with T cells had
higher concentrations of NO. By adding iNOS inhibitor L-NIL
to the SSC low CD11b + Gr1 dim cells and T cells co-culture, T cell
proliferation was restored. Conclusion: SSC low CD11b + Gr1 dim
cells represent authentic monocytic MDSCs in the liver, and
their numbers were increased in the livers of NAFLD mice. The
suppressive function of monocytic MDSCs was dependent on
NO production by iNOS. These cells might have a role in the
pathogenesis of NAFLD.
Disclosures:
The following authors have nothing to disclose: Liying Yao, Masanori Abe, Teruki
Miyake, Yoshiko Nakamura, Yusuke Imai, Yohei Koizumi, Takao Watanabe,
Osamu Yoshida, Masashi Hirooka, Yoshio Tokumoto, Bunzo Matsuura, Yoichi
Hiasa