Levosimendan up-regulates transforming growth factor-beta and smad signaling in the aorta in the early stage of sepsis.

Abstract

This prospective, controlled experimental study was planned to investigate the effects of levosimendan on transforming growth factor (TGF)-beta3 and Smad1, Smad2 and Smad3 expression in the early stages of sepsis.
Twenty-four rats were randomized into four groups: (1) sham-operated controls, (2) dobutamine group--subjected to abdominal hypertension and peritonitis-induced sepsis using cecal ligation and puncture (CLP), then treated with 10 microg x kg(-1) min(-1) intravenous (IV) dobutamine infusion, (3) levosimendan group--as in 2, then treated with levosimendan IV bolus 200 microg x kg(-1) followed by 200 microg x kq(-1) min(-1) IV infusion, and (4) a control group as in 2, with no treatment. All rats were killed 8 hours after CLP. Aorta tissue samples were analyzed by immunohistochemical staining.
CLP caused mild interleukin (IL)-1 immunostaining in both control and dobutamine groups. Immunoreactivity of tumor necrosis factor (TNF)-alpha was mild in both sham and control groups. TGF-beta3 immunostaining was mildly increased in groups sham, control and dobutamine, whereas it was found moderate in group levosimendan. Smad1, Smad2 and Smad3 were found moderately increased only in group levosimendan.
Beneficial effects of levosimendan on hemodynamics and global oxygen transport were reported in experimental and clinical trials. Besides its potency on C++ ion sensitivity, it should influence inflammatory cytokine production by diminishing TGF-beta3 and Smad1, Smad2 and Smad3 expression.

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293
Turkish Journal of Trauma & Emergency Surgery
Experimental StudyDeneysel Çalışma
Ulus Travma Acil Cerrahi Derg 2010;16 (4):293-299
Levosimendan up-regulates transforming growth factor-beta
and Smad signaling in the aorta in the early stage of sepsis
Levosimendan erken dönem sepsiste aortada “transforming growth factor beta” ve
Smad işaretlenmesini up-regüle eder
Koray ERBÜYÜN,1 Demet TOK,1 Seda VATANSEVER,2 Gülay OK,1
Elgin TÜRKÖZ,2 Hasan AYDEDE,3 Yamaç ERHAN,3 İdil TEKİN1
Departments of 1Anesthesiology, 2Histology and Embryology,
3General Surgery, Celal Bayar University, Faculty of Medicine,
Manisa, Turkey.
Celal Bayar Üniversitesi Tıp Fakültesi, 1Anesteziyoloji Anabilim Dalı,
2Histoloji ve Embriyoloji Anabilim Dalı, 3Genel Cerrahi Anabilim Dalı,
Manisa.
Correspondence (İletişim): Koray Erbüyün, M.D. Celal Bayar Üniversitesi Tıp Fakültesi, Anesteziyoloji ve Reanimasyon Anabilim Dalı, 45040 Manisa, Turkey.
Tel: +90 - 232 - 373 80 38 Fax (Faks): +90 - 236 - 237 02 13 e-mail (e-posta): korayerbuyun@yahoo.com
AMAÇ
Bu prospektif kontrollü deneysel çalışma, levosimendanın
erken dönem sepsiste transforming growth factor (TGF)-β3
ve Smad1, Smad2 ve Smad3 oluşumuna etkilerini araştır-
mak amacıyla planlandı.
GEREÇ VE YÖNTEM
Yirmi dört sıçan rasgele dört gruba ayrıldı: 1) sham kont-
rol grubu 2) dobutamin grubu - abdominal hipertansiyon,
çekal ligasyon ve delme (ÇLD) tekniği ile peritonit mode-
li prensibiyle sepsis oluşturuldu, sonra sıçanlar 10 µg.kg-1
dk-1 intravenöz (IV) dobutamin infüzyonu ile tedavi edil-
di, 3) levosimendan grubu - 2. grupta olduğu gibi sepsis
modeli gerçekleştirildi sonra sıçanlar levosimendan IV bo-
lus 200 µg.kg-1 takiben, 200 µg.kg.-1 dk-1 IV infüzyonu ile
tedavi edildi, 4) kontrol grubundakilere tedavi yapılmadı.
Tüm sıçanlar ÇLD’den 8 saat sonra öldürüldü. Aort doku
örnekleri immünohistokimyasal olarak boyandı.
BULGULAR
ÇLD hem kontrol, hem de dobutamin gruplarında ılım-
lı IL-1 boyanmasına neden oldu. Tumor necrosis factor
(TNF)-α immünreaktivitesi sham ve kontrol grubunda
ılımlıydı. Sham, kontrol ve dobutamin gruplarında TGF-β3
ılımlı derecede yüksek bulunurken, bu parametre levos-
mendan grubunda orta derecede yüksek saptandı. Smad1,
Smad2 ve Smad3 levosmendan grubunda orta derecede
yükselmiş olarak saptandı.
SONUÇ
Levosimendanın hemodinami ve global oksijen taşınması-
na sağladığı olumlu etkiler, deneysel ve klinik çalışmalarda
bildirilmiştir. Levosimendanın C++ iyon duyarlılığı üzerine
etkileri yanı sıra, TGF-β3, Smad1, Smad2 ve Smad3 oluşu-
munu etkileyerek enflamasyonda sitokin salınımına da et-
kileri olabilir.
Anahtar Sözcükler: Levosimendan; sepsis; Smad; transforming
growth factor-β.
BACKGROUND
This prospective, controlled experimental study was
planned to investigate the effects of levosimendan on
transforming growth factor (TGF)-β3 and Smad1, Smad2
and Smad3 expression in the early stages of sepsis.
METHODS
Twenty-four rats were randomized into four groups: 1)
sham-operated controls, 2) dobutamine group - subjected
to abdominal hypertension and peritonitis-induced sepsis
using cecal ligation and puncture (CLP), then treated with
10 µg.kg-1min-1 intravenous (IV) dobutamine infusion,
3) levosimendan group - as in 2, then treated with
levosimendan IV bolus 200 µg.kg-1 followed by 200 µg.kg.-1
min-1 IV infusion, and 4) a control group as in 2, with no
treatment. All rats were killed 8 hours after CLP. Aorta
tissue samples were analyzed by immunohistochemical
staining.
RESULTS
CLP caused mild interleukin (IL)-1 immunostaining in
both control and dobutamine groups. Immunoreactivity
of tumor necrosis factor (TNF)-α was mild in both sham
and control groups. TGF-β3 immunostaining was mildly
increased in groups sham, control and dobutamine, where-
as it was found moderate in group levosimendan. Smad1,
Smad2 and Smad3 were found moderately increased only
in group levosimendan.
CONCLUSION
Beneficial effects of levosimendan on hemodynamics and
global oxygen transport were reported in experimental and
clinical trials. Besides its potency on C++ ion sensitivity, it
should influence inflammatory cytokine production by di-
minishing TGF-β3 and Smad1, Smad2 and Smad3 expres-
sion.
Key Words: Levosimendan; sepsis; Smad; transforming growth
factor-β.

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294 Temmuz - July 2010
Ulus Travma Acil Cerrahi Derg
Uncontrolled release of inflammatory molecules
such as nitric oxide, cytokines, reactive oxygen spe-
cies and endothelins precede major changes in hemo-
dynamics and tissue hypoperfusion, contributing to
multiorgan failure. Support of tissue perfusion with
adequate blood pressure and organ blood flow is a
crucial component of sepsis treatment. Management
of sepsis-induced circulatory failure aims at restoring
cardiac output and arterial blood pressure by fluid re-
suscitation and inotrope/vasopressor administration.[1]
Inadequate tissue perfusion persists even after effec-
tive volume replacement[2] and vasoactive therapy.[3]
The use of a new calcium sensitizer, levosimendan,
in patients with septic shock was first described in a
case report in 2005,[4] and since then, case reports,[5]
case series,[6] randomized controlled trials[7,8] and ex-
perimental studies[9-11] have reported improvements in
hemodynamics, global oxygen transport, pulmonary
circulation, metabolism vasopressor requirements,
and tissue blood flow and oxygenation.
Abdominal compartment syndrome (ACS) is an
important complication for septic patients, and as a
cause of abdominal hypertension, ACS has adverse ef-
fects on the circulation, threatening the function and
viability of tissues.[12,13] Pathophysiologic effects in-
clude release of cytokines, formation of oxygen free
radicals, and decreased cellular production of adenos-
ine triphosphate, leading to the translocation of bac-
teria from the gut and intestinal edema predisposing
to multiorgan dysfunction syndrome that affects vital
body systems.
We previously demonstrated that proinflammatory
cytokine (tumor necrosis factor (TNF)-α, interleukin
(IL)-1β) production was diminished in a levosimen-
dan-treated cecal ligation and puncture (CLP)-induced
sepsis experimental model,[14] and we aimed to inves-
tigate if transforming growth factor-β3 (TGF-β3) and
R-Smads (Smad1, Smad2, Smad3) were affected.
In the present study, we complicated the CLP-in-
duced sepsis model with abdominal hypertension to
mimic the clinical situation of sepsis, and our hypothe-
sis is that levosimendan should attenuate inflammation
by up-regulating TGF-β3 and R-Smads in the early
stages of sepsis. To test this hypothesis, we investi-
gated and compared the immunohistochemical stain-
ing of aorta tissue in the early stage of an experimen-
tal model of peritonitis-induced sepsis. We compared
levosimendan with dobutamine because systemic and
regional tissue perfusion effects of these two drugs had
been investigated and compared in some experimen-
tal endotoxemia models previously,[15] but the effects
of these two drugs on the aorta in peritonitis-induced
early stages of sepsis has not been studied.
MATERIALS AND METHODS
The study was conducted in the Experimental Ani-
mal Research Laboratory of University Hospital and
approved by the local ethical committee. The study
conformed to the recommendations of the Helsinki
Declaration. Twenty-four male adult Wistar albino rats
weighing 200-250 g were housed in plastic cages in a
controlled environment with free access to water and
food.
Anesthesia and Preparation
All animals were anesthetized with intramuscular
(IM) 75 mg.kg-1 ketamine and one femoral artery was
catheterized. For measurement of mean arterial blood
pressure (MAP), the arterial catheter was connected
to a pressure transducer (Sasan pressure set, Sasan
Medical Disposable Products, Mönchengladbach,
Germany). MAP was monitored continuously (Da-
tex Engstrom Ohmeda, Finland) and recorded every
15 minutes (min). The contralateral femoral vein was
catheterized for fluid administration. A saline infusion
was started as described in the study protocol and ad-
justed to maintain the MAP within 10 mmHg of initial
levels.
Cecal Ligation and Puncture - Abdominal
Hypertension
The rats’ peritoneal cavities were opened and CLP
was performed according to a previously standardized
technique described by Wichterman and colleagues.
[15] The cecum was devascularized, ligated in a way
that preserves intestinal continuity and perforated in a
standardized fashion with an 18-gauge needle in two
locations on the antimesenteric surface and left open.
Then, two 20 gauge catheters were placed in two sides
of the abdominal cavity parallel to each other and their
tips were kept outside the abdominal cavity. Subse-
quently, the laparotomy incision was closed in two
layers. The catheters were connected to a sphygmoma-
nometer’s pump, and intraabdominal pressure (IAP)
was increased in a 5 min period by means of air pump-
ing, until an intraperitoneal pressure of 15 mmHg
was obtained. This level of IAP was sustained for the
whole experiment with air pumping on demand.
Study Design
The surgical preparation was followed by 30 min
of stabilization and then the animals were randomized
into four equal groups. All rats were killed eight hours
after stabilization.
Group S (Sham): Femoral arterial and venous
catheterization was done, intraabdominal catheters
were placed, but IAP was not elevated. MAP was
monitored; 20 mL.kg-1h-1 saline infusion was started.
Group C (Control): Femoral arterial and venous
catheterization was done, CLP was applied, intraab-

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Cilt - Vol. 16 Sayı - No. 4295
Levosimendan up-regulates TGF-β and Smad signaling in the aorta in the early stage of sepsis
dominal catheters were placed, MAP was monitored,
and 20 mL.kg-1 h-1 saline infusion was started. Four
hours later, the intravenous (IV) saline infusion rate
was increased to 30 mL.kg-1 h-1 and the IAP was el-
evated to 15 mmHg.
Group D (Dobutamine): Treated as in C, with the
addition of 10 µg.kg-1 min-1 IV dobutamine (Dobuta-
mine hydrochloride 250 mg.20mL-1, Mayne Pharma,
Mulgrave, Australia) infusion at the 4-hour time point,
which continued during the study.
Group L (Levosimendan): Treated as in C, with
the addition of 200 µg.kg-1 levosimendan (Simdax 2.5
mg.mL-1, Abbott Laboratories, Abbott Park, IL, USA)
as an IV bolus begun at the 4-hour time point, and con-
tinued as a 200 µg.kg-1 min-1 IV infusion during the
study.
Histological Evaluation
Tissue collection and fixation: Samples from all
groups were fixed in 10% formalin solution for up to
48 hours (h). They were then washed with tap water
and embedded in paraffin following a routine embed-
ding procedure. Cross-sections of 5 μ were taken from
the blocks and prepared for both histochemical and
immunohistochemical staining.
Histochemical Observation
Sections dewaxed under 60 ºC overnight were im-
mersed in xylene for one hour and then rehydrated
through a graded series of ethanol (100%, 95%, 80%,
70%, and 60%) for 2 min in each concentration and
they were then washed in tap water. Sections were
stained with either hematoxylin-eosin (H-E) accord-
ing to routine protocols. Slides were mounted using
Entellan, covered with glass cover slips prior to view-
ing, and photographed under the Olympus BX-40 (To-
kyo, Japan) light microscopy.
Immunohistochemical Staining
The sections were incubated at 60 ºC overnight
and then dewaxed in xylene for 30 min. After soak-
ing in a decreasing series of ethanol, sections were
washed with distilled water. They were then treated
with 2% trypsin in 50 mM Tris buffer (pH 7.5) at 37
ºC for 15 min, and washed with phosphate-buffered
saline (PBS). Sections were delineated with an Elite
Pap pen (DBS, Pleasanton, CA, USA) and incubated
in 3% H2O2 solution for 15 min to inhibit endogenous
peroxidase activity. They were washed three times for
5 min with PBS and incubated with primary antibod-
ies, TGF-β3 (sc-82, Santa Cruz, CA, USA), Smad1,
Smad2, Smad3 (sc-7960, Santa Cruz, CA, USA), IL-1
β (sc-7884, Santa Cruz, CA, USA), and TNF-α (sc-
7317, Santa Cruz, CA, USA) for 18 h. Next, the sec-
tions were incubated with biotinylated IgG (supplied
ready to use) for 30 min, followed by three washes in
PBS and then with streptavidin-peroxidase conjugate
(supplied ready to use) for 30 min (KP-500, Universal
Phosphatase Kit, Diagnostic BioSystems, Pleasanton,
CA, USA) and washed with PBS three times. They
were then incubated with a solution containing diami-
nobenzidine (DAB, 1718096, Roche, Mannheim, Ger-
many) 50 µL for each section for 5 min to visualize
immunolabeling, and after rinsing with distilled water,
counterstained with Mayer’s hematoxylin (72804E,
Microm, Walldorf, Germany). The sections were de-
hydrated with 80% and 95% alcohol and immersed in
xylene and covered with mounting media (H701, CC/
Mount, Universal Phosphatase Kit, Diagnostic Bio-
Systems, Pleasanton, CA, USA). The negative con-
trols received the same treatment as described above,
but were incubated with rabbit IgG or mouse IgG in-
stead of the primary antibodies.
Immunolabeling intensity was graded indepen-
dently by two observers blinded to the experimental
conditions with the following scale: mild (+), moder-
ate (++) and strong (+++).
Data Analysis
The software SPSS 10.0 for Windows (SPSS, Chi-
cago, IL, USA) was used for statistical evaluation. All
values are expressed as mean±SEM. Differences in
MAP among groups were statistically analyzed with
one-way ANOVA. A p value of <0.05 was considered
as significant.

RESULTS
No mortality was encountered in any of the groups
8 h after CLP. There was no difference in MAP during
the study period in all groups (Fig. 1); these data were
taken from our previously published study.[14] Immu-
nohistochemical staining results are summarized in
Table 1.
CLP caused mild increase in IL-1β immunostain-
ing in both C and D groups (Fig. 2). Immunoreactiv-
ity of TNF-α was mildly increased in both S and C
groups (Fig. 3). TGF-β3 immunostaining was mild in
Groups S, C and D, whereas it was found moderate-
ly increased in Group L (Fig. 4). Smad1, Smad2 and
Smad3 immunostaining was found moderate only in
Group L (Fig. 5).
Fig. 1. Mean arterial pressures. No significant difference was
seen between the groups.
80
Group S
Group C
Group L
Group D
Time
MAP (mmHg)
85
90
95
100
105
110

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Ulus Travma Acil Cerrahi Derg
296 Temmuz - July 2010
DISCUSSION
The effects of levosimendan on transforming
TGF-β3 and R-Smads in the aorta during the early
stages of sepsis in rats were investigated in the pres-
ent study. The ability of levosimendan to reduce pro-
inflammatory cytokines and in addition to up-regulate
TGF-β3 and R-Smads was demonstrated, while dobu-
tamine had no beneficial effect.
We used a peritonitis model with CLP that produc-
es vasodilatation and increased cardiac output similar
to early stages of human sepsis,[16] which mimics the
clinical situation. The early hyperdynamic state of
septic patients corresponds well with the hemodynam-
ic situation seen in rats after initiation of CLP.[15,17,18] In
contrast, lipopolysaccharide (LPS) models often gen-
erate a situation with a profound hypodynamic state.
[19] In the present study, during the first eight hours of
CLP, hemodynamic variables did not change as severe
as in the LPS-induced sepsis models and the macro-
circulation was comparable between all of the treated
groups. This enabled the model to test the effects of
dobutamine and levosimendan on tissue injury by
eliminating the detrimental effects of arterial hypo-
tension and myocardial depression induced by endo-
toxemia and the specific effects of these drugs on the
myocardium and vascular tonus.
Abdominal hypertension worsens physiologic pa-
rameters including hemodynamics, abdominal perfu-
sion, pulmonary functions, and survival.[12,13] Second-
ary ACS is an important risk for patients with sepsis
who are treated with large volumes of infused resus-
citation fluids, which leads to decreased abdominal
wall compliance, capillary leakage and bowel edema.
Rezende-Neto et al.[20] showed that intraabdominal hy-
pertension stimulated the production of pro-inflamma-
tory agents, and even after abdominal decompression,
neutrophil accumulation and inflammatory infiltration
continued. In the present study, we added abdominal
hypertension to a well-known model of peritonitis in
order to simulate the clinical picture of patients having
intra-abdominal hypertension in addition to sepsis.
Case reports and observational studies suggest ben-
eficial effects of levosimendan in septic patients,[4,7]
and preemptive use of levosimendan before the induc-
tion of endotoxemia in pigs prevented the development
of hypodynamic endotoxic shock.[21] Levosimendan
Table 1. Immunolabeling intensity of IL-1β, TNF-α, Smad1, Smad2, Smad3
and TGF-β3


Group S
(Sham)
Group C
(Control)
Group D
(Dobutamine)
Group L
(Levosimendan)
IL-1β
TNF-α
Smad 1/2/3
TGF-β3
– +
+
–
+
–/+
–
–
+
–
––/+
–
+
++
++
Mild (+), moderate (++) and strong (+++).
Fig. 2. IL-1β immunostaining.
A: Group S (DAB x 40), B: Group
C (DAB x 100), C: Group L (DAB
x 40), D: Group D (DAB x 100).

Page 5

improved hemodynamics, global oxygen transport,
pulmonary circulation, and right ventricular function
in septic shock patients with acute respiratory distress
syndrome.[8] Data from the available clinical studies
suggest that rational use of levosimendan is a safe and
effective alternative to increasing dobutamine doses
in treating the failing heart of septic shock patients.
[7,8] Levosimendan exerts its positive inotropic effect
by increasing contractile myofilament sensitivity to
C++ ions. Besides its potency on C++ ion sensitivity,
we speculated whether it would influence inflamma-
tory cytokine production. Thus, we previously inves-
tigated the proinflammatory cytokine (TNF-α, IL-1β)
production in dobutamine- and levosimendan-treated
rats during the early phase of CLP-induced sepsis, and
we found that levosimendan could diminish the pro-
inflammatory cytokine production in lung tissue.[14]
In the present study, we aimed to investigate whether
TGF-β3 and R-Smads would play a role in this anti-
inflammatory process.
TGF-β3 is a multifunctional cytokine with a broad
spectrum of effects on cell differentiation, prolifera-
tion, and activation of immune cells, and it is impli-
cated in immune abnormalities linked to cancer, au-
toimmunity, opportunistic infections, and fibrotic
complications.[22]
TGF- β appears to play a key regulatory role in lim-
Levosimendan up-regulates TGF-β and Smad signaling in the aorta in the early stage of sepsis
Cilt - Vol. 16 Sayı - No. 4 297
Fig. 3. TNF-α immunostaining.
A: Group S (DAB x 100), B: Group
C (DAB x 100), C: Group L (DAB
x 100), D: Group D (DAB x 100).
Fig. 4. TGF-β3 immunostaining.
A: Group S (DAB x 40), B: Group
C (DAB x 100), C: Group L (DAB
x 100), D: Group D (DAB x 40).