studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 383A
340
Novel Vasodilator and Anti-inflammatory Drug-based
Strategy for Restoring Ischemia/ reperfusion-Exposed
Donor Liver to Healthy State
Thirunavukkarasu Chinnasamy 1,2 , Fadi L. Jaber 1 , Sanjeev Gupta 3 ;
1 Department of Medicine, Albert Einstein College of Medicine,
Bronx, NY, USA, Bronx, NY; 2 Department of Biochemistry and
Molecular Biology, Pondicherry University, Puducherry, India;
3 Departments of Medicine and Pathology, Marion Bessin Liver
Research Center, Diabetes Center, Cancer Center, Ruth L. and
David S. Gottesman Institute for Stem Cell and Regenerative Medicine
Research, Albert Einstein College of Medicine, Bronx, NY
Maintaining donor organs in healthy state is critical for isolating
viable cells and for liver transplantation. A major cause of
donor organ deterioration is ischemia/reperfusion (IR) injury
due to complex events, e.g., vasoconstriction or inflammation
with release of ROS, cytokines/chemokines/receptors,
etc. We hypothesized that donor organ preconditioning with
early control of vasoconstriction and inflammation by NO and
TNF-α blockade as master regulators of interest will be effective.
Therefore, we used NO donor, nitroglycerin (NG), and
TNF-α blocker, Etanercept (ETN), before nonlethal hepatic IR in
F344 rats with clamping of portal and hepatic blood flow for
15 min and reperfusion for 24h. In drug-untreated controls, IR
produced much oxidative stress, including hepatic GGT expression,
nitrotyrosine, 8-oxo-dG DNA adducts and liver necrosis,
as expected. In hepatocytes isolated from donors with IR, cell
viability and attachment in dishes was inferior to control donor
cells without IR. However, cultured cells from IR donors better
withstood secondary H 2
O 2
or CCl 4
injury. By contrast, preconditioning
with ETN 12-18h before I/R and/or NG starting
5min before IR and continuing during and after IR for 20 min
lessened hepatic injury. ETN+NG combination was most effective
versus either drug alone in restoring IR liver to near-normal
without necrosis or GGT and nitrotyrosine expression. Hepatocytes
from ETN+NG-preconditioned rats with IR were more
viable, attached better in dishes, and were also more sensitive
to H 2
O 2
and CCl 4
injury, which was similar to healthy control
hepatocytes. FACS analysis showed that polyploid hepatocytes
were depleted in cells from IR rats but not in cells from healthy
control and ETN+NG preconditioned donors, again indicating
less oxidative insult. Next, to further examine potential of
isolated donor cells, we performed cell transplantation assays
in DPPIV- rats. Hepatocytes from healthy donor rats engrafted
and were normally distributed in periportal areas in zones
1/2 of liver lobules. However, hepatocytes from IR donor rats
engrafted less and were mainly in portal vein radicles, suggesting
differences in their cell adhesion and matrix-interacting
properties. By contrast, engraftment and intrahepatic distribution
of transplanted hepatocytes isolated from ETN+NG preconditioned
livers was similar to cells from healthy control rats.
Conclusions: The donor liver was successfully preconditioned
and restored to normal health with a simple regimen of NO-donor
and TNF-α blockade. This drug approach will be readily
translated to clinical setting for improving outcomes in liver
transplantation and use of donor livers for cell therapy.
Disclosures:
The following authors have nothing to disclose: Thirunavukkarasu Chinnasamy,
Fadi L. Jaber, Sanjeev Gupta
341
Identification of microRNAs Associated with Allograft
Tolerance
Matthew J. Vitalone 1 , Liang Wei 2 , Karine Piard-Ruster 1 , Carlos
O. Esquivel 1 , Olivia M. Martinez 1 , Sheri M. Krams 1 ; 1 Surgery,
Stanford University School of Medicine, Stanford, CA; 2 Sichuan
Academy of Medical Sciences and Sichuan Provincial People’s
Hospital, Chengdu, China
Introduction: Although the liver is less immunogenic than
other solid organs, liver transplant recipients receive lifelong
immunosuppression. The aim of this study was to profile the
expression of microRNAs (miRNAs) associated with the induction
and maintenance of tolerance to an allograft. Methods:
Previous studies showed that donor-specific tolerance can be
induced in recipients of rat orthotopic liver transplants (OLT)
after post-transplant total lymphoid irradiation (TLI). To identify
miRNAs associated with tolerance we profiled liver grafts from
syngeneic (DAàDA) and allogeneic OLT recipients (DAàLewis)
that received post-transplant TLI, allograft recipients that were
not treated post-transplant, and normal DA livers. Untreated
allograft recipients reject their grafts within 10 days whereas
TLI treated recipients have long-term graft survival (>100 days),
thus miRNAs were examined at two time points, seven days
(induction of tolerance) and 100 days (established tolerance)
post-transplant Results: A supervised principal component analysis
(PCA) using the top 15 differentially altered miRNA was
able to robustly model the study groups with as little as three
principal components describing 88.5% of the variance of
the data. Indeed, three miRNA, miR142-3p, miR142-5p and
miR181a were determined to be key miRNA associated with
tolerance. Further, analysis of the full complement of miRNAs,
by PCA, revealed that both the established tolerance and the
syngeneic groups occupied the same relative three-dimensional
space close to the normal liver group, indicating similar miRNA
profiles. Conclusions: A small group of miRNA can distinguish
graft status post-transplant. Further, our findings support a
model whereby tolerant liver allografts express a profile consistent
with that of healthy livers.
Disclosures:
The following authors have nothing to disclose: Matthew J. Vitalone, Liang Wei,
Karine Piard-Ruster, Carlos O. Esquivel, Olivia M. Martinez, Sheri M. Krams
342
Previous ischemic preconditioning in experimental
model of steatotic liver trasplantation with brain death
Mónica B. Jiménez-Castro 1 , Mariana Mendes-Braz 1 , Jordi
Gracia-Sancho 3 , Maria Eugenia Cornide-Petronio 1 , Araní
Casillas-Ramírez 2 , Juan Rodes 1 , Carmen Peralta 1 ; 1 Institut D’Investigacions
Biomèdiques August Pi i Sunyer, Barcelona, Spain; 2 Hospital
Regional de Alta Especialidad de Ciudad Victoria, Ciudad
Victoria, Mexico; 3 Barcelona Hepatic Hemodynamic Laboratory,
IDIBAPS, CIBEREHD, Institut D’Investigacions Biomèdiques August
Pi i Sunyer, Barcelona, Spain
Background & Aims: Liver transplantation has evolved to
become a standard therapy for certain end-stage liver diseases.
Nowadays, high percent of organs come from donors who
have suffered brain trauma-brain dead donors, which may also
show hepatic steatosis, being both characteristics risk factors
in liver transplantation. Nevertheless brain dead reduces the
tolerance of liver grafts to the preservation/reperfusion injury
and reduces graft survival. Ischemic preconditioning shows
benefits when applied in the liver transplantation from nonbrain
dead patients like hepatectomies, whereas it has been
less promising in the transplantation from brain dead patients.