fk330, a novel inducible nitric oxide synthase inhibitor, prevents ischemia and reperfusion injury...

American Journal of Transplantation 2006; 6: 2013–2022Blackwell Munksgaard

C© 2006 The AuthorsJournal compilation C© 2006 The American Society of

Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2006.01435.x

FK330, a Novel Inducible Nitric Oxide SynthaseInhibitor, Prevents Ischemia and ReperfusionInjury in Rat Liver Transplantation

S. Tsuchihashi,a F. Kaldas,a N. Chida,b

Y. Sudo,b K. Tamura,c Y. Zhai,a B. Qiao,a

R. W. Busuttila and J. W. Kupiec-Weglinskia,∗

aThe Dumont-UCLA Transplant Center, Division of Liverand Pancreas Transplantation, Department of Surgery,David Geffen School of Medicine at UCLA, Los Angeles,California, USAbPharmacology Research Laboratories, Astellas PharmaInc., Osaka, JapancAstellas Research Institute of America, Evanston, Illinois,USA∗Corresponding author: Jerzy W. Kupiec-Weglinski,[email protected]

Nitric oxide (NO), produced via inducible NO synthase(iNOS), is implicated in the pathophysiology of liverischemia/reperfusion injury (IRI). We examined the ef-fects of a novel iNOS inhibitor, FK330 (FR260330), inwell-defined rat liver IRI models. In a model of liver coldischemia followed by ex vivo reperfusion, treatmentwith FK330 improved portal venous flow, increasedbile production and decreased hepatocellular damage.FK330 prevented IRI in rat model of 40-h cold ischemiafollowed by syngeneic orthotopic liver transplantation(OLT), as evidenced by: (1) increased OLT survival (from20% to 80%); (2) decreased hepatocellular damage(serum glutamic oxaloacetic transaminase/glutamicpyruvic transaminase levels); (3) improved histologi-cal features of IRI; (4) reduced intrahepatic leukocyteinfiltration, as evidenced by decreased expression ofP-selectin/intracellular adhesion molecule 1, ED-1/CD3cells and neutrophils; (5) depressed lymphocyte activa-tion, as evidenced by expression of pro-inflammatorycytokine (TNF-a , IL-1b , IL-6) and chemokine (IP-10,MCP-1, MIP-2) programs; (6) prevented hepatic apop-tosis and down-regulated Bax/Bcl-2 ratio. Thus, bymodulating leukocyte trafficking and cell activationpatterns, treatment of rats with FK330, a specific iNOSinhibitor, prevented liver IRI. These results provide therationale for novel therapeutic approaches to maxi-mize organ donor pool through the safer use of livergrafts despite prolonged periods of cold ischemia.

Key words: Ischemia/reperfusion, liver, nitric oxide,transplant

Received 20 March 2006, revised 14 April 2006 andaccepted for publication 1 May 2006

Introduction

Liver transplantation has been proven as a successful treat-

ment for end-stage liver disease. However, primary graft

nonfunction or early dysfunction may occur, significantly af-

fecting both morbidity and mortality. Ischemia/reperfusion

injury (IRI), an antigen-independent inflammatory compo-

nent of organ procurement, remains an important factor

in poor graft function. It causes up to 10% of early liver

transplant failure, and can lead to the higher incidence of

acute and chronic rejection (1). The mechanism of IRI in-

volves microcirculatory flow disturbances caused by en-

dothelial cell (EC) adhesion, leukocyte tethering with sub-

sequent sequestration in tissue, activation of Kupffer cells

leading to the release of pro-inflammatory cytokines and

chemokines, as well as the formation of reactive oxygen

species (ROS) and reactive nitrogen species (RNS), result-

ing in sinusoidal EC death and ultimately in hepatocyte

damage (1–8).

Nitric oxide (NO) is a gaseous molecule produced from

the amino acid L-arginine by nitric oxide synthase (NOS)

(9). NOS can be classified into two categories: constitutive

NOS, which includes neuronal NOS (nNOS) and endothe-

lial NOS (eNOS), and inducible NOS (iNOS) (10). It has been

suggested that NO produced by endothelial, eNOS, may be

an important endogenous protective molecule that limits

the tissue injury caused by IRI (11,12). In contrast, exces-

sive amounts of NO produced from iNOS is detrimental

(11,13–15). Extensive production of NO may lead to ox-

idative damage by interacting with ROS such as superox-

ide anion produced by macrophages, polymorphonuclear

leukocytes (PMNs), and EC. Interaction between superox-

ide anion and NO may lead to the formation of peroxynitrite,

a cytotoxic molecule capable of initiating lipid peroxidation,

and nitrotyrosine, resulting in loss of protein structure and

function (4). It has been shown that nonselective inhibition

of NOS can exacerbate damage secondary to IRI by de-

creasing eNOS-dependent NO (16,17). However, selective

iNOS inhibition may be beneficial (13,14,18). From these

observations, it is clear that NO induced by iNOS can be

cytotoxic in pathophysiology.

FK330 (FR260330), is a newly developed specific iNOS in-

hibitor with a novel mechanism of action that inhibits iNOS

monomer dimerization and selectively blocks enzyme

2013

Tsuchihashi et al.

activity (19). Recently, the effective dose of FK330 to in-

hibit NO production, and its efficacy to prevent chronic aor-

tic graft rejection in rats (20) and renal IRI in monkeys (21)

have been shown. The aim of this study was to evaluate

putative cytoprotective effects of FK330 in rat liver models

of cold ischemia followed by ex vivo reperfusion or in vivo

syngeneic orthotopic liver transplantation (OLT).

Materials and Methods

AnimalsMale Sprague-Dawley (SD) rats (200–250 g; Harlan Sprague-Dawley, Inc.,

Indianapolis, IN, USA) were housed in the UCLA animal facility under spe-

cific pathogen-free condition. Animals received humane care according to

the criteria outlined in the “Guide for the Care and Use of Laboratory Ani-

mals” prepared by the National Academy of Sciences and published by the

National Institutes of Health (NIH publication 86–23 revised 1985).

FK330FK330 (FR260330) was supplied by Astellas Pharma Inc. (Osaka, Japan).

FK330 was suspended in 0.5% methylcellulose (Fisher Scientific Interna-

tional Inc., IL, USA). Oral suspension (4 mg/mL) was prepared extempora-

neously before administration.

Model of liver cold ischemia followed by ex vivo reperfusionAfter skeletonization of the liver, the portal vein, bile duct and inferior vena

cava were cannulated and the liver was flushed with 10 mL of University of

Wisconsin (UW) solution. After 30-h storage at 4◦C in UW, livers were per-

fused with rat whole blood diluted with Krebs-Ringer bicarbonate medium

to a hematocrit of 15% on an isolated perfusion rat liver apparatus for 2 h,

with stable temperature (37◦C), pressure (13 cm H2O) and pH (7.4), as de-

scribed (7). There were two experimental groups (n = 8 rats/group). Group I:

livers and blood for ex vivo perfusion were obtained from untreated rats.

Group II: livers were recovered from rats that were pre-treated with FK330

(20 mg/kg p.o.) 90 min prior to liver procurement; blood donors also re-

ceived FK330 (20 mg/kg p.o. at −90 min). During ex vivo perfusion, portal

vein blood flow and pressure were recorded every 15 min, while bile output

was monitored every 30 min. Blood was collected at 30 min intervals. At the

conclusion of experiment, liver tissue was fixed in formalin for histological

evaluation.

Syngeneic OLT modelLivers from SD rats were stored at 4◦C in UW for defined periods prior

to being transplanted into syngeneic recipients with revascularization but

without hepatic artery reconstruction. There were two major OLT groups.

Group I: livers from untreated rats were stored for 30 h (n = 6), 40 h (n =10) or 48 h (n = 6) at 4◦C, and then transplanted into otherwise untreated

recipients. Group II: OLT hosts received FK330 (20 mg/kg p.o. at −90 min)

and then twice daily (40 mg/kg/day) for 3 days (n = 10); donor rats were

also treated with FK330 (20 mg/kg p.o. at −90 min). OLT recipients were

followed for survival. Separate rat groups (n = 6) were killed at 6 h, and OLT

samples were collected for analysis. All microsurgery experiments were

performed by the very same operator.

Hepatocellular functionTo assess hepatocellular function in ex vivo and in vivo hepatic cold IRI mod-

els, serum glutamic oxaloacetic transaminase (sGOT) and glutamic pyruvic

transaminase (sGPT) levels were measured using an autoanalyzer (ANTECH

Diagnostics, Irvine, CA, USA).

HistologyLiver specimens were fixed in 10% buffered formalin solution and embed-

ded in paraffin. Sections were made at 5 lm and stained with hematoxylin

and eosin. The blindly assessed histological severity of liver IRI was graded

using modified Suzuki’s criteria (22). In this classification, sinusoidal conges-

tion, hepatocyte necrosis and ballooning degeneration are graded from 0

to 4. No necrosis, congestion or centrilobular ballooning is given a score of

0, while severe congestion and ballooning degeneration, as well as greater

than 60% lobular necrosis is given a value of 4.

ImmunohistologySnap frozen liver samples were processed, as described (7,8). Primary

antibodies (Abs) against P-selectin, (CD62P; Pharmingen, San Diego,

CA, USA), intracellular adhesion molecule 1 (ICAM-1; Pharmingen),

ED-1 (macrophage/monocyte; CHEMICON International, Inc., Temecula,

CA, USA), CD3 (T cell; Pharmingen) were added at optimal dilutions. Bound

primary Abs were detected using peroxidase-conjugated goat anti-rabbit

or anti-mouse secondary Abs (Dako Envision kit/HRP). Negative controls

included sections in which the primary Ab was replaced with dilution

buffer or normal mouse serum. The sections were developed with 3,3′-diaminobenzidine tetrahydrochloride, and evaluated blindly by counting la-

beled cells in triplicate in 10 high-power fields/section.

RNA extraction and real-time RT-PCRTotal RNA was extracted from the liver using the TRIzol reagent (Life Tech-

nologies, Inc., Grand Island, NY, USA). The mRNA coding for iNOS, TNF-a,

IL-1b, IL-6, IP-10, MCP-1, MIP-2 and b-actin was quantified in triplicate by

real-time RT-PCR, as described (23). The sequences of the primers are listed

in Table 1. Thermal cycling conditions were 10 min at 95◦C followed by

40 cycles of 95◦C for 15 s and 60◦C for 1 min on an ABI PRISM 7000 Se-

quence Detection System (Applied Biosystems, Foster City, CA, USA). Each

gene expression was normalized to b-actin mRNA content and calculated

relative to naı̈ve control using the comparative CT method.

Western blotsLiver samples were homogenized, and separated on 5–20% polyacry-

lamide gels, as described (7,8). The membranes were incubated with

specific primary Ab against iNOS, eNOS (BD Transduction Laboratories,

CA, USA), Bcl-2 (Abcam Inc., Cambridge, MA, USA), Bax (Cell Signaling

Technology, Inc., Beverly, MA, USA) and b-actin (Abcam Inc.), followed

by horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Pierce

Biotechnology, Rockford, IL, USA) secondary Ab. Immunoreactive bands

were visualized by SuperSignal West Pico Chemiluminescent Substrate

(Pierce Biotechnology). Relative quantities of protein were determined by

densitometer (Kodak Digital Science 1D Analysis Software, Rochester, NY,

USA) and presented in comparison to b-actin.

NO synthesisNO production was determined by measuring serum nitrite and nitrate lev-

els using a commercially available kit (Cayman Chemical, Ann Arbor, MI,

USA).

Myeloperoxidase assayThe presence of myeloperoxidase (MPO), an enzyme specific for neu-

trophils, was used as an index of intrahepatic neutrophil accumulation, as

described (7). One unit of MPO activity was defined as the quantity of en-

zyme degrading 1 lM peroxide/min at 25◦C per gram of tissue.

Detection of apoptosisApoptosis was detected using the terminal deoxynucleotidyl transferase-

mediated dUTP nick end-labeling (TUNEL) method, as described (8). The

2014 American Journal of Transplantation 2006; 6: 2013–2022

FK330 in Liver Ischemia and Reperfusion Injury

Table 1: Sequences of the primers for SYBR green real-time RT-PCR

Target genes Forward primers Reverse primers

iNOS 5′-GGTCTTTGAAATCCCTCCTGA-3′ 5′-AGCTCCTGGAACCACTCGTA-3′TNF-a 5′-CGTAGCCCACGTCGTAGC-3′ 5′-GGTTGTCTTTGAGATCCATGC-3′IL-1b 5′-GCTGACAGACCCCAAAAGAT-3′ 5′-AGCTGGATGCTCTCATCTGG-3′IL-6 5′-CCTGGAGTTTGTGAAGAACAACT-3′ 5′-GGAAGTTGGGGTAGGAAGGA-3′IP-10 5′-CCAACCTTCCAGAAGCACCAT-3′ 5′-ACCGTTCTTGCGAGAGGGAT-3′MCP-1 5′-GATCTCTCTTCCTCCACCACTATG-3′ 5′-GAATGAGTAGCAGCAGGTGAGT-3′MIP-2 5′-ACTCTTTGGTCCAGAGCCATG-3′ 5′-TGGTAGGGTCGTCAGGCATT-3′b-actin 5′-TGCCAACACAGTGCTGTCTG-3′ 5′-GAGCCACCAATCCACACAGAG-3′

results were scored semi-quantitatively by averaging the number of

TUNEL+ cells/field. Six-fields/tissue sample were evaluated.

Statistical analysisData are expressed as mean ± SEM. Differences between the groups were

analyzed using one-way analysis of variance or Student’s t-test for unpaired

data. For the survival study, Kaplan–Meier log-rank analysis was performed.

All differences were considered statistically significant at the p-value of

<0.05.

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Figure 1: Effects of FK330 in rat livers perfused for 2 h on the isolated perfusion apparatus after 30 h of cold ischemia. (A)

FK330 treatment significantly increased portal blood flow throughout the perfusion period, as compared with controls (∗p < 0.05). (B) Bile

production at 30-min intervals throughout the reperfusion period was also significantly higher in the group treated with FK330 as compared

with controls (∗p < 0.05). (C) The sGOT levels were significantly lower at 90 and 120 min during the perfusion of the livers treated with

FK330, as compared with controls (∗p < 0.005). (D) Circulating NO levels throughout the reperfusion period. FK330 treatment significantly

decreased serum NO levels, as compared with controls (∗p < 0.001). These data represent mean ± SEM of eight independent perfusions

for each group.

Results

FK330 treatment in rat liver model of cold ischemiafollowed by ex vivo reperfusionWe monitored portal vein blood flow, bile production, and

sGOT levels in livers that were recovered from untreated or

FK330 pre-treated SD rats, stored for 30 h at 4◦C in UW so-

lution, and then perfused for 2 h on the isolated perfusion

rat liver apparatus with syngeneic whole blood obtained

American Journal of Transplantation 2006; 6: 2013–2022 2015

Tsuchihashi et al.

from untreated or FK330 pre-treated donors. FK330 treat-

ment significantly improved portal vein blood flow through-

out the reperfusion period (Figure 1A; p < 0.05), increased

bile production (Figure 1B; p < 0.05), and decreased sGOT

levels (Figure 1C; p < 0.005), as compared with untreated

livers. The efficacy of FK330 was confirmed when serum

nitrite and nitrate, the stable inactive end products of the

NOS pathway, were measured. Unlike in controls, FK330

significantly decreased circulating NO levels throughout

the reperfusion period (p < 0.01). This therapeutic effect re-

quired combined liver graft and blood donor treatment with

FK330. Treatment of liver donors alone or blood donors

alone with FK330 failed to ameliorate I/R-induced hepato-

cellular damage (data not shown).

We graded hepatocellular injury by the Suzuki’s histol-

ogy criteria. In the control group, livers showed severe

sinusoidal and vascular congestion with marked vacuoliza-

tion change, focally associated with hepatocyte necrosis

(Figure 2A; Suzuki’s score = 6.2 ± 0.7). In contrast, FK330

treatment group revealed minimal hepatocyte necrosis,

and significantly less sinusoidal congestion, as compared

with controls (Figure 2B; Suzuki’s score = 1.2 ± 0.3, p <

0.001).

FK330 treatment in rat liver model of cold ischemiafollowed by OLTFK330 prolongs OLT survival, improves hepatic func-tion and ameliorates hepatocellular injury: We next ex-

amined whether FK330 conferred protection against hep-

atic IRI in vivo. SD livers were recovered from donor rats

that remained untreated or were pre-treated with FK330.

Livers were then stored at 4◦C for 30, 40 or 48 h before

being transplanted into syngeneic recipients that remained

untreated or received FK330. As shown in Figure 3, the an-

ex vivo

Co

ntr

ol

FK

330

OLT

A

B

C

D

Figure 2: Photomicrographs of rep-resentative rat livers after 30 h coldischemia, and 2 h of reperfusion onthe isolated perfusion rat liver ap-paratus or OLT recovered at 6 h. (A)

Control group with sinusoidal/vascular

congestion and severe lobular distor-

tion (Suzuki’s score = 6.2 ± 0.7).

(B) FK330-treated group with minimal

vascular congestion/vacuolar degener-

ation and preservation of lobular archi-

tecture (Suzuki’s score = 1.2 ± 0.3, p <

0.001). (C) Control OLTs group present

extensive areas of necrosis and si-

nusoidal/vascular congestion (Suzuki’s

score = 8.3 ± 0.5). (D) FK330 OLTs

group present minimal vascular con-

gestion, necrosis and less lobular dis-

tortion (Suzuki’s score = 1.8 ± 0.5; p <

0.001 compared with controls). (×100,

hematoxylin and eosin stain; n = 4–

6/group).

imal survival for 30 h cold-stored OLT was 100% in both

untreated control and FK330 groups. FK330 treatment sig-

nificantly prolonged 14-day animal survival from 20% (2/10)

to 80% (8/10) in OLT that were cold-stored for 40 h (p <

0.002). In contrast, only 17% (1/6) of recipients survived

after 48 h of liver cold storage with or without FK330.

The prolonged survival after FK330 treatment in the 40 h

cold-stored OLTs correlated with improved liver function,

as measured by sGOT/GPT levels (1294 ± 94/749 ± 195

and 5783 ± 878/3448 ± 589 IU/L in FK330 and control OLT,

respectively; p < 0.01; Figure 4).

The 40 h cold-stored OLT were assessed using the Suzuki’s

histological classification. The control group showed mod-

erate to severe hepatocyte necrosis with disruption of

lobular architecture and marked sinusoidal congestion

(Figure 2C; Suzuki’s score = 8.3 ± 0.5). In contrast, the

FK330 treated group showed minimal necrosis and no

evidence of sinusoidal congestion (Figure 2D; Suzuki’s

score = 1.8 ± 0.5; p < 0.001).

FK330 reduces intrahepatic iNOS and serum NO levels:The iNOS gene and protein expression, as determined by

real-time PCR and Western blots, respectively, were signif-

icantly increased in untreated control OLT at 6 h, as com-

pared with naı̈ve controls (Figure 5A and 5B). The iNOS

protein somewhat decreased at 24 h and became unde-

tectable at day 3 post-OLT. FK330 treatment significantly

decreased iNOS mRNA/protein expression (p < 0.001). In

contrast to iNOS, there was no difference in the eNOS

protein expression between untreated control and FK330

groups (Figure 5B). Significantly increased serum NO levels

found during untreated hepatic IRI as compared to naı̈ve

rats, were markedly reduced following FK330 treatment

(Figure 5C).



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40h CIT FK330 (n=10)


48h CIT FK330 (n=6)


30h CIT FK330 (n=6)

Day after OLT

Figure 3: Effects of FK330 on OLTsurvival. Donor rat livers (FK330 pre-treated or untreated) were storedfor 30 to 48 h at 4◦C in UW solution,and then transplanted to groupsof FK330 treated or untreated re-cipients. In the 40 h cold-preserved

OLT, FK330 treatment significantly pro-

longed the animal survival from 20% to

80% at 2 weeks post-OLT (p < 0.002; n

= 10 rats/group). OLT = orthotopic liver

transplantation; CIT = cold ischemic

time.

sG

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(IU

/L)

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Figure 4: Serum transaminase lev-els (IU/L) after 40 h of cold ischemiafollowed by OLTs. Levels of sGOT and

sGPT were significantly lower at 6 h in

the FK330 treatment groups compared

with untreated controls (∗p < 0.005).

These data represent mean ± SEM of

six independent experiments.

FK330 modulates leukocyte trafficking and prevents in-trahepatic cell infiltration: Figure 6 shows immunohis-

tochemical staining for P-selectin (A and B) and ICAM-1 (C

and D), the molecules that mediate adhesive leukocyte–

endothelium interactions at the inflammatory site. At 6 h

after reperfusion higher expression of P-selectin and ICAM-

1 were observed in OLT of untreated control recipients

(Figure 6A and 6C), as compared with FK330-treated group

(Figure 6B and 6D, respectively). To determine whether

FK330 treatment affected local leukocyte infiltration, we

measured macrophage (ED-1)/T-cell (CD3) infiltration by im-

munohistochemical staining and PMN infiltration by MPO

assay. Indeed, FK330 treatment markedly decreased intra-

graft infiltration of ED-1 (Figure 6F) and CD3 (Figure 6H)

positive cells, as compared with controls (Figure 6E and

6G). In addition, MPO activity in OLT of FK330-treated

group was decreased as compared with controls (1.10 ±0.15 vs. 3.00 ± 0.32; p < 0.01; data not shown), suggesting

diminished neutrophil infiltration.

FK330 decreases pro-inflammatory cytokine andchemokine expression: As shown in Figure 7, FK330

treatment significantly decreased intragraft expression of

mRNA coding for macrophage-associated TNF-a , IL1b and

IL-6, as compared with control OLTs (p < 0.05). Further-

more, FK330 reduced the expression of chemokines (IP-

10, MCP-1 and MIP-2), as compared with controls (p <

0.05).

FK330 promotes anti-apoptotic function: TUNEL as-

say was carried out to examine whether FK330 treat-

ment has an anti-apoptotic effect on hepatic IRI. At 6 h

after transplantation, the number of TUNEL positive cells

was markedly decreased in the FK330 group (3.6 ± 0.6;

Figure 8B and 8C), as compared with untreated controls

(26.6 ± 3.1; p < 0.0001; Figure 8A and 8C).

We then used Western blots to analyze the expres-

sion of anti-apoptotic Bcl-2 and pro-apoptotic Bax pro-

teins. The densities of protein bands for Bax or Bcl-2

were quantitated and the ratio of Bax to Bcl-2 was cal-

culated. As shown in Figure 9, significantly up-regulated

Bax to Bcl-2 ratio was recorded in control OLTs, as com-

pared with naı̈ve livers (1.47 ± 0.05 vs. 0.56 ± 0.07;

p = 0.0006). FK330 treatment down-regulated the Bax

to Bcl-2 ratio (0.89 ± 0.07; P = 0.0007, as compared

with controls) suggesting FK330-mediated anti-apoptotic

function.


Tsuchihashi et al.

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Figure 5: Effect of FK330on iNOS/eNOS expres-sion and NO produc-tion in rat livers coldstored for 40 h followedby OLT. (A) Real-time RT-

PCR-assisted iNOS mRNA

levels. Means ± SEM

are shown; n = 4/group.∗p = 0.03. (B) Western

blot-assisted iNOS/eNOS

protein levels. Means ±SEM are shown; n =4/group. ∗p = 0.001. (C)

Serum NO levels. Means

± SEM are shown; n =4/group. ∗p < 0.001.

P-selectin ICAM-1

Co

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FK

330

ED-1 CD3

A C

B D

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Figure 6: Immunohist-ochemical staining forP-selectin, ICAM-1, ED-1and CD3 in rat liversrecovered at 6 h aftertransplantation. Treat-

ment with FK330 de-

creased expression of

P-selectin (B), ICAM-1

(D), ED-1 (F) and CD3 (H)

positive cells, as com-

pared with controls (A, C,

E and G). Representative

of three OLTs is shown.

(Original magnification,

×400).

Discussion

We report here the results of our studies on the protective

effects of targeting iNOS with FK330 (FR260330), a novel

specific iNOS inhibitor, in rat liver models of cold ischemia

followed by ex vivo reperfusion or syngeneic OLT. In the

ex vivo hepatic cold IRI model, FK330 pre-treatment sig-

nificantly improved portal venous flow, increased bile pro-

duction, diminished neutrophil infiltration and decreased

hepatocellular damage. FK330 therapy in OLT recipients:

(1) improved liver function and hepatocyte integrity with re-

sultant prolongation of graft survival from 20% to 80%; (2)

decreased the expression of P-selectin and ICAM-1; (3) se-

lectively prevented neutrophil, T-cell and macrophage infil-

tration; (4) inhibited pro-inflammatory cytokine/chemokine

expression and (5) reduced hepatic apoptosis in parallel

with decreased Bax/Bcl-2 ratio. These cytoprotective ef-

fects, which required treatment of both donor and recipi-

ent rats with FK330, were correlated with the inhibition of

iNOS-induced NO production.



TNF- IL-1β

IP-10 MCP-1 MIP-2

IL-6

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* * *

*

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α

Figure 7: Real-time RT-PCR-assisted expressionof pro-inflammatory cy-tokine and chemokinegenes at 6 h after OLTs.Treatment with FK330 de-

creased the expression of

TNF-a, IL-1b, IL-6, IP-10,

MCP-1 and MIP-2, as com-

pared with controls (∗p <

0.05). Each graph repre-

sents the mean ± SEM

of four independent exper-

iments.

Control FK330

C

A B

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Figure 8: TUNEL-assisted detectionof apoptotic cells in rat OLTs at 6 h af-ter transplantation. The frequency of

TUNEL positive cells in control group

(A and C; 26.6 ± 3.1) was markedly

increased, as compared with those in

FK330 group (B and C; 3.6 ± 0.6, ∗p

< 0.001). A minimum of 6 fields was

evaluated per sample (magnification of

×200); n = 3.

NO, a free radical or RNS, is a versatile mediator, the effects

of which can either contribute to or counteract pathological

processes in IRI. eNOS is constitutively expressed in en-

dothelium under basal conditions and the low level of NO

produced regenerates hepatic perfusion, prevents platelet

adhesion, thrombosis, PMN accumulation and secretion of

inflammatory mediators (11,12). NO also induces vasodila-

tion at the level of the sinusoid and at pre-sinusoid sites to

keep a balance with vasoconstrictors such as endothelin.

iNOS has not been found to be constitutively present in the

normal liver but can be induced by pro-inflammatory media-

tors, such as cytokines and lipopolysaccharide or during I/R,

shock, trauma and infection. Induction of iNOS may have

either toxitic or protective effects, depending on the type

of insult, the level and duration of iNOS expression and

the simultaneous production of superoxide anion. Kaizu


Tsuchihashi et al.

Bax

Bcl-2

β-Actin

20-

28-

42-

kDa Naïve Control FK330

00.20.40.60.81.01.21.41.61.8

Naïve Control FK330

Ba

x/B

cl-

2

*

Figure 9: Western blot analysis ofBax and Bcl-2 in rat OLTs. Control

OLTs significantly up-regulated the ra-

tio of Bax to Bcl-2, as compared with

naı̈ve liver (p = 0.0006). FK330 treat-

ment significantly down-regulated the

Bax to Bcl-2 ratio, as compared with

controls (∗p = 0.0007). The densities

of protein bands for Bax or Bcl-2 were

quantitated and the ratio of Bax to Bcl-

2 was calculated. These data represent

mean ± SEM of four independent ex-

periments.

et al. (24) has shown that donor pre-treatment with ade-

noviral iNOS significantly up-regulated intragraft iNOS ex-

pression and ameriolated liver IRI with reduced iNOS in-

duction ratio in cold-preserved rat OLT. Their results sug-

gest that I/R-induced iNOS exerts cytotoxic, whereas iNOS

pre-conditioning results in cytoprotective functions in liver

IRI. Although excessive NO synthesized by iNOS has been

indicated as an important mediator in the development of

IRI, inhibition of iNOS or iNOS deficiency reduced IRI in

liver (13), heart (25), kidney (14,21) and intestine (18,26).

Hepatic iNOS expression has been observed during early

phase of IRI. Yagnik et al. (17) has shown that liver iNOS

protein expression peaked at 6 h post-reperfusion and de-

creased by 12 to 24 h in cold-preserved rat OLT. In our

study, the most prominent iNOS protein expression was

also observed at 6 h and decreased at 24 h post-OLT. The

increased iNOS expression triggered by organ recovering

itself (Tsuchihashi, unpublished) may explain the need for

FK330 to treat both blood and liver donors in this study.

Moreover, extended graft preservation can induce strong

iNOS expression after reperfusion. van der Hoeven et al.

(15) have shown that 40 h cold preservation significantly

increased graft iNOS expression, which led to reduced sur-

vival, as compared with 30 h preserved OLTs. Indeed, in the

present study 40 h cold-preserved control OLTs showed

significantly increased iNOS gene/protein and serum NO

levels at 6 h of reperfusion. FK330 treatment effectively

prevented liver IRI as evidenced by decreased sGOT/GPT,

improved histological features such as lobular ballooning,

hepatocyte necrosis, sinusoidal congestion, and prolonged

OLT survival. In the ex vivo liver IRI model, FK330 treatment

improved the portal blood flow, increased bile production

and reduced sGOT levels. In both models, reduced iNOS

expression and/or NO production were strongly correlated

with cytoprotection rendered by FK330. In contrast, FK330

did not affect eNOS expression in OTLs. FK330 is a selec-

tive iNOS inhibitor believed to act by binding to the oxyge-

nase domain of the inactive iNOS monomer, thereby pre-

venting formation of the active iNOS dimer. Indeed, in our

previous in vitro study, FK330 exhibited potent inhibitory

activity against cytokine-induced iNOS activity in human

colon cancer cells (DLD-1) (19). Collectively, these results

suggest that excessive NO production may accelerate liver

IRI and that inhibition of NO induced by iNOS is an impor-

tant pharmacological effect of FK330.

Reoxygenation of the ischemic liver causes generation of

numerous ROS and RNS. Although low concentrations

of ROS/RNS have an important role as mediators in nor-

mal cellular metabolism and signal transduction (27,28), in

higher concentrations they can be damaging due to con-

comitant consumption of endogenous antioxidants, pro-

duction of inflammatory cytokines and apoptotic or necrotic

cell death.

Both ROS and RNS can activate Kuppfer cells, which

leads to production of cytokines such as TNF-a and IL-

1b (29,30), which may then exert direct cytotoxic effects

on endothelium cells and hepatocytes (4,5,31). Hepatic IRI

significantly induced the expression of pro-inflammatory



TNF-a, IL-1b and IL-6 in this study. However, FK330 treat-

ment effectively reduced these cytokine, which other-

wise up-regulate the expression of adhesion molecules fa-

voring leukocyte–sinusoidal EC interactions and triggering

cytokine cascades. Leukocyte–EC interactions are well

established as playing a role in the pathophysiology of

hepatic IRI (7,8). Initial leukocyte tethering in sinusoidal

EC requires expression of CD62 selectins. In sinusoidal

EC, P-selectin and ICAM-1 become up-regulated by I/R

through ROS/RNS (5,32–34). NO, a free radical or RNS

can activate this inflammatory cascade and thus promote

the development of IRI. We have previously shown that

CD62 blockade with recombinant P-selectin glycoprotein

ligand-immunoglobulin fusion protein reduces IRI after rat

liver (35,36) and intestinal (37) transplantation. Cuzzocrea

et al. (38) have reported that intestinal ischemia followed

by reperfusion for 4 h resulted in the up-regulation of P-

selectin and ICAM-1. They also observed that significant

reduction of P-selectin and ICAM-1 expressions in iNOS

KO mice and wild-type counterparts treated with a se-

lective iNOS inhibitor which reduced NO production, cy-

tokines (TNF-a, IL-1b and IL-6) expression and neutrophil

infiltration. Indeed, in our study, FK330 treatment inhib-

ited serum NO levels and markedly reduced intrahepatic

expression of iNOS, adhesion molecules (P-selectin and

ICAM-1), and cytokines (TNF-a, IL-1b and IL-6)/chemokines

(IP-10, MIP-2 and MCP-1). Intrahepatic infiltration of leuko-

cytes, including PMNs (MPO assay), macrophages (Ed-1)

and T cells (CD3), were all markedly suppressed in FK330-

treated OLTs, as compared with controls. These data sug-

gest that iNOS-induced NO modulates the expression of

cytokines/chemokines and adhesion molecules and sub-

sequent leukocyte migration and infiltration, which in turn

prevents local microcirculatory disturbances and develop-

ment of primary graft dysfunction.

I/R-induced NO or its byproducts, peroxynitrite, may pro-

mote apoptosis through multiple mechanisms including

cytochrome c release through mitochondrial membrane

permeability, direct DNA damage and signaling pathways

involving MAP kinase (39,40). NO increases Bax pro-

tein expression and triggers increase in Bax to Bcl-2 ra-

tio resulting in increased mitochondrial permeability and

cytochrome c release (41). The induced release of cy-

tochrome c and complete dissipation of the inner mito-

chondrial membrane as well as collapse of oxidative phos-

phorylation lead to accelerated formation of large amounts

of ROS with deleterious consequences (42). This may rep-

resent an important mechanism by which NO induces

apoptosis in IRI. Wu et al. (43) have shown that the ac-

tivation of iNOS after I/R enhances mucosal apoptosis

in the rat small intestine, which is due to the release

of cytochrome c from mitochondria and the inhibition of

iNOS ameliorated apoptosis after IRI. Indeed, FK330 treat-

ment in this study significantly decreased the number of

apoptotic cells and Bax/Bcl-2 ratio in OLTs. These results

suggest that NO mediates cell apoptosis by increasing

Bax/Bcl-2 ratio and that inhibition of iNOS may have an

important anti-apoptotic and protective effect in hepatic

IRI.

In conclusion, targeting iNOS with a selective novel in-

hibitor, FK330, protected against hepatic IRI. The block-

ade of iNOS-induced NO prevented local inflamma-

tory/apoptotic responses by modulating cell trafficking and

reducing neutrophil, macrophage and T-cell infiltration. This

resulted in the inhibition of pro-inflammatory cytokines and

chemokines, and reduced Bax/Bcl-2 ratio. Thus, treatment

with FK330 is an effective strategy, which provides the ra-

tionale for novel therapeutic approaches to maximize the

organ donor pool through the safer use of liver grafts de-

spite prolonged periods of cold ischemia.

Acknowledgment

This work was funded by the NIH Grants RO1 DK062357, AI23847 and

AI42223 (J.W.K.W), and the Dumont Research Foundation.

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