JPET #124057 1 Anti-eosinophilic activity of simendans ...jpet.aspetjournals.org/content/jpet/early/2007/07/09/jpet.107.124057.full.pdfJPET #124057 4 Abstract Simendans are novel agents

JPET #124057 1

Anti-eosinophilic activity of simendans

Hannu Kankaanranta, Xianzhi Zhang, Ritva Tumelius, Minna Ruotsalainen, Heimo Haikala,

Erkki Nissinen, Eeva Moilanen

The Immunopharmacology Research Group, Medical School, FIN-33014 University of

Tampere (HK, XZ, EM) and Department of Respiratory Medicine (HK) and Research Unit

(EM), Tampere University Hospital, Tampere, Finland, Seinajoki Central Hospital, Finland

(HK), The Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of

Sciences, Beijing, China (XZ), Orion Corporation, Espoo, Finland (RT, MR, HH, EN)

JPET Fast Forward. Published on July 9, 2007 as DOI:10.1124/jpet.107.124057

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on July 9, 2007 as DOI: 10.1124/jpet.107.124057

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Running title: anti-eosinophilic activity of simendans

Correspondence to: Hannu Kankaanranta, MD, PhD

Department of Respiratory Medicine

Seinajoki Central Hospital

Hanneksenrinne 7

FIN-60220 Seinajoki, FINLAND

Tel: +358 6 415 4849

Fax: +358 6 415 4989

e-mail: [email protected]

Number of text pages: 35

Number of tables: 2

Number of figures: 7

Number of references 41

Number of words in abstract: 188

Number of words in Introduction 490

Number of words in Discussion 1288

List of non-standard abbreviations

Ann-V : annexin-V

BAL : bronchoalveolar lavage

[Ca2+]i : intracellular free calcium concentration

Dextrosimendan : (+)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-

pyridazinyl)phenyl)hydrazono]propanedinitrile


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GM-CSF : granulocyte-macrophage colony-stimulating factor

IL : interleukin

JNK : c-jun-N-terminal kinase

KATP : ATP-sensitive potassium channel

Levosimendan : (-)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-

pyridazinyl)phenyl)hydrazono]propanedinitrile

L-JNKI1 : GRKKRRQRRR-PP-RPKRPTTLNLFPQVPRSQD-amide

L-TAT : RKKRRQRRR-amide; negative control for L-JNKI1

PD098059 : 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one

PI : propidium iodide

SB203580 : 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4 pyridyl)-1H-

imidazole

Z-Asp-CH2-DCB : benzyloxycarbonyl-Asp-CH2COC-dichlorobenzene

A recommended section:

Inflammation, Immunopharmacology and Asthma


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Abstract

Simendans are novel agents used in the treatment of decompensated heart failure. They

sensitise troponin C to calcium and open ATP sensitive potassium channels (KATP), and have

been shown to reduce cardiac myocyte apoptosis. The aim of the present study was to

evaluate whether simendans reduce pulmonary eosinophilia and regulate eosinophil apoptosis.

Bronchoalveolar lavage (BAL) eosinophilia was evaluated in ovalbumin-sensitized mice.

Effects of simendans on apoptosis in isolated human eosinophils were assessed by relative

DNA fragmentation assay, annexin-V-binding, and morphological analysis. Dextrosimendan

reduced ovalbumin-induced BAL-eosinophilia in sensitised mice. Levosimendan and

dextrosimendan reversed interleukin-5 (IL)- afforded survival of human eosinophils by

inducing apoptosis in vitro. Even high concentrations of IL-5 were not able to overcome the

effect of dextrosimendan. Dextrosimendan further enhanced spontaneous apoptosis as well as

that induced by CD95 ligation, without inducing primary necrosis. Dextrosimendan-induced

DNA fragmentation was shown to be dependent on caspase and JNK activation, whereas

extracellular signal-regulated kinase, p38 mitogen- activated kinase and ATP sensitive

potassium channels seemed to play no role in its actions. Taken together, our results show that

simendans possess anti-eosinophilic activity and may be useful for the treatment of

eosinophilic inflammation.


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Introduction

Eosinophils are thought to play a critical role in allergic diseases, such as allergic rhinitis,

atopic dermatitis and asthma (Gleich, 2000). In asthmatic patients, activation of eosinophils in

the airways is thought to cause epithelial tissue injury, contraction of airway smooth muscle

and increased bronchial responsiveness. Apoptosis or programmed cell death may be an

important feature in the resolution of asthmatic inflammation (Kankaanranta et al., 2005).

Apoptosis is characterized by specific biochemical and morphological changes, including cell

shrinkage, surface blebbing, DNA fragmentation and loss of nucleoli (Kankaanranta et al.,

2005), so that the apoptotic cell is phagocytosed intact without release of its contents.

Eosinophil apoptosis is inhibited by cytokines IL-3, IL-5 and GM-CSF (Kankaanranta et al.,

2005), whereas eosinophil apoptosis is up-regulated by Fas (CD95/APO-1), a 45 kDa

transmembrane protein belonging to the tumor necrosis factor receptor family (Kankaanranta

et al., 2005). We and others have previously shown that eosinophil apoptosis is delayed in

patients with asthma or inhalant allergy (Wedi et al., 1997; Kankaanranta et al., 2000).

Furthermore, the number of eosinophils in asthmatic lung is elevated and is inversely

correlated with the number of apoptotic eosinophils (Vignola et al., 1999).

Levosimendan is a novel agent with a dual mechanism of action developed and

marketed for the treatment of decompensated heart failure. This agent sensitises troponin C to

calcium in a manner dependent on calcium concentration, thereby increasing the effects of

calcium on cardiac myofilaments during systole and improving contraction at low energy cost

(Pollesello et al., 1994; Haikala et al., 1995). Levosimendan causes also vasodilatation

through the opening of ATP sensitive potassium channels (Yokoshiki et al., 1997). In addition

to its beneficial effects in decompensated heart failure (Follath et al., 2002), levosimendan has

been shown to have beneficial effects in left ventricular failure complicating acute myocardial

infarction by lowering the risk of death (Moiseyev et al., 2002). This suggests that


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levosimendan may affect the inflammatory events associated with myocardial infarction in

addition to its beneficial effects on hemodynamics. In fact, supporting the possible anti-

inflammatory effects, levosimendan has been shown to have beneficial effects in carrageenan-

induced paw edema in rats (Haikala H et al., 2004) and in experimentally-induced septic

shock in pigs (Oldner et al., 2001), to affect the inflammatory changes associated with

decompensated advanced heart failure in humans (Parissis et al., 2004) and to reduce cardiac

myocyte apoptosis in rats (Louhelainen et al., 2007).

Given the possible role of Ca2+ and K+ in eosinophil apoptosis (Beauvais et al.,1995;

Bankers-Fullbright et al., 1998; Beauvais et al., 1998) our aim was to test the effects of

simendans (Figure 1) on pulmonary eosinophilia and on human eosinophil apoptosis. The

present study describes the ability of dextrosimendan to reduce pulmonary eosinophilia in

ovalbumin-sensitized mice and that simendans are able to induce apoptosis in human

eosinophils and to reverse IL-5-afforded eosinophil survival as well as evaluates their possible

mechanisms of action. We describe dextrosimendan as a novel anti-inflammatory drug

candidate with anti-eosinophilic properties.


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Materials and Methods

Animals

BALB/c mice 6 to 8 weeks old (20-25 g) were purchased from M&B A/S (Ry, Denmark).

The mice were housed in the animal facility of OrionPharma Ltd. in a thermostatically

controlled room at 22 ± 2 °C at a relative humidity of 40-70 % with artificial illumination

from 6 a.m. to 8 p.m.. Mice were fed commercial pellets (SDS rat and mouse no 1

maintenance, Special Diet Services Ltd., UK) and given water ad libitum under specific

pathogen-free conditions according to standard guidelines. All animal experiments were

conducted with the permission of the Animal Ethics Committee of OrionPharma, in

accordance with Finnish law and government regulations complying with the European

Community guidelines for the use of experimental animals.

Allergen sensitization and exposure

The mice were sensitized by initial i.p. injection of ovalbumin (20 µg in 2 mg Al(OH)3/0.2 ml

0.9% NaCl) on day 0. After 1 week, these mice were further sensitized with the same reagent.

On days 21, 22 and 23, the mice were exposed to aerosols of allergen (1% ovalbumin in 0.9%

NaCl) for 20 minutes on each day. The aerosol challenge was performed in a sealed plastic

chamber by using a jet nebulizer (Hugo Sachs Elektronik, Germany) at the pressure 1.5 bar.

Animal treatments and BAL analysis

Sensitized and challenged animals were treated with vehicle (2.5 ml/kg of 0.1% Tween-20 in

0.9% NaCl), dextrosimendan (0.1 and 1.0 mg/kg) or budesonide (1.0 mg/kg) intranasally 1 h

before and 6 h after challenge at days 21-23. Twenty four hours after last challenge animals

were euthanized with an overdose of pentobarbital sodium (180 mg/kg i.p.). Bronchoalveolar

lavage (BAL) was collected by washing lungs twice with 0.5 ml of PBS (+37oC). Each wash


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included gentle flush for 3 times. Volume of BAL fluid was measured and filled into 1 ml,

and the number of white blood cells was determined by a hemocytometer Sysmex Microcell

Counter (TOA Medical Electronics, Japan). Differential cell counts were calculated from

May-Grünwald-Giemsa-stained cytospin slides (white blood cell number 2x105/slide).

Human eosinophil purification

Blood (50–100 ml) was obtained from eosinophilic individuals. Eosinophils were isolated to

> 99% purity under sterile conditions as previously reported (Kankaanranta et al., 1999; 2000,

Zhang et al., 2000). The cells were resuspended at 106 cells /ml and cultured in RPMI 1640,

10% fetal calf serum and antibiotics. Subjects gave informed consent to a study protocol

approved by the ethics committee of Tampere University Hospital (Tampere, Finland).

Determination of eosinophil apoptosis

Unless otherwise stated, eosinophil apoptosis was determined by propidium iodide staining of

DNA fragmentation and flow cytometry as previously described (Kankaanranta et al., 1999;

2000, 2006; Zhang et al., 2000). The cells showing decreased relative DNA content were

considered to be apoptotic. Annexin-V binding and morphological analysis was performed as

previously reported (Zhang et al., 2002). Oligonucleosomal DNA fragmentation in

eosinophils was analyzed by agarose gel DNA electrophoresis as previously described

(Kankaanranta et al., 1999; 2006; Zhang et al., 2003). Eosinophil apoptosis is expressed as

apoptotic index (number of apoptotic cells / total number of cells).


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Immunoblot analysis

Eosinophils were suspended at 106 cells/ ml and cultured at 37 oC. At the indicated time

points, cells were centrifuged at 12000 g for 10 min. The cell pellet was lysed by boiling for 5

min in 30 µl of Laemmli buffer (x 6), centrifuged at 12000 g and debris was carefully

removed. Samples were then stored at –20 oC until analyzed. For immunoblot analysis, each

protein sample was loaded on 8-12% SDS-PAGE gel and electrophoresed for 2 h at 100 V.

The separated proteins were transferred to nitrocellulose membrane (HybondTM

ECLTM

) with

semi-dry blotter, blocked using 5% non-fat dry milk in TBS/T. Proteins were labelled using

specific antibody and subsequently detected using Super-Signal™ West Dura Extended

Duration substrate (Pierce, Rockford, IL, USA) western blotting detection agents and detected

by using Fluorchem 8800 equipment and software (AlphaInnotec, San Leandro, CA, USA).

Quantification of relevant bands was performed by densitometry. The activated JNK was

identified and quantified by western blot analysis using specific antibody recognizing the dual

phosphorylated (i.e. activated) form of JNK. Control time curve with the solvent (0.5%

DMSO) was prepared to see the change in JNK activation in similar conditions in the absence

of dextrosimendan. The increase in activation of JNK by dextrosimendan is expressed as the

phospho-JNK activity in dextrosimendan-treated cells as compared with the simultaneously

prepared solvent control cells.

Materials

Levosimendan and dextrosimendan were obtained from OrionPharma Ltd., Espoo, Finland.

The synthesis of racemic simendans and isolation of levo- and dextrosimendan has been

described earlier (Haikala et al., 1990, Nore et al., 1992). Reagents were obtained as follows:

L-JNKI1 and L-TAT control peptide (Alexis Corp, Läufelfingen, Switzerland), Z-Asp-CH2-


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DCB (Peptide Institute, Inc, Osaka, Japan), phosphospecific JNK monoclonal antibody (Santa

Cruz Biotechnology, Santa Cruz, CA, USA), horseradish peroxide-linked anti-rabbit IgG

(Amersham Life Science, Amersham, UK), and budesonide, diazoxide, glibenclamide, 5-

hydroxydecanoate and ovalbumin (Sigma Chemical Co, St. Louis, MO). Unless otherwise

stated, the reagents were obtained as previously described (Kankaanranta et al., 1999; 2000;

2002; 2006; Zhang et al., 2000; 2002; 2003). The incubation time was 40 h unless otherwise

stated. L-JNK1, L-TAT, PD098059, SB203580 and Z-Asp-CH2-DCB were added 20 min

before dextrosimendan. Stock solutions of levo-/dextrosimendan, PD098059, SB203580 and

Z-Asp-CH2-DCB were prepared in DMSO. Budesonide was dissolved in ethanol. The final

concentration of DMSO in the culture was 0.5 % and that of ethanol 0.2%. A similar

concentration of the solvent was added to the control cultures.

Statistics

Results are expressed as means ± S.E.M. Statistical significance was calculated by Welch t-

test or analysis of variance for repeated measures supported by Dunnett or Student-Newman-

Keuls test. Differences were considered significant if P < 0.05.


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Results

Effect of dextrosimendan on ovalbumin-induced lung eosinophilia in sensitized mice

In non-sensitized animals the total number of eosinophils in BAL fluid after challenge was

very low (0.001 ± 0.001 x105 eosinophils accounting to 0.06 + 0.04 % of all cells in BAL;

n=17). Sensizitisation with ovalbumin dramatically increased the number of eosinophils in

BAL-fluid after challenge (2.87 ± 0.65 x105 eosinophils) accounting to 44 ± 3% of all cells in

BAL. Treatment of animals with dextrosimendan (0.1 or 1 mg /kg) reduced the number of

eosinophils in BAL as compared with vehicle treated animals in a dose-dependent manner

(Figure 2). For comparison, treatment of animals with budesonide (1 mg/kg) almost

completely prevented eosinophilia in BAL (2 ± 0.5 % eosinophils in BAL; n=20; P<0.001 as

compared with the sensitized and challenged control animals).

Effects of levo- and dextrosimendan on IL-5-afforded eosinophil survival

During culture for 40 hours, IL-5 inhibited human eosinophil apoptosis in a concentration-

dependent manner. Maximal inhibition of apoptosis was obtained at 10 pM concentration of

IL-5 (apoptotic indexes 0.57 ± 0.09 and 0.07 ± 0.02 in the absence and presence of IL-5,

respectively, n=5, P<0.001). Levosimendan increased the number of apoptotic eosinophils in

the presence of 10 pM IL-5 (ie. reversed the effect of IL-5), with an EC50 value of 6.5 ± 0.7

µM (Figure 3A). This increase in the number of apoptotic cells was confirmed by showing

increased phosphatidylserine expression on the outer leaflet of cell membrane of IL-5-treated

cells, i.e. the percentage of Annexin-V-positive cells in the absence and presence of

levosimendan (10 µM) was 7±1% and 86±4%, respectively. Dextrosimendan reversed IL-5-

afforded human eosinophil survival in a concentration-dependent manner by inducing

apoptosis (EC50 2.9 ± 0.4 µM; Figure 3B). The ability of dextrosimendan to induce apoptosis

in IL-5-treated human eosinophils was confirmed by showing the increase in the percentage


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of Annexin-V positive eosinophils (5 ± 1% and 95 ± 1% in the absence and presence of 40

µM dextrosimendan, respectively, n=6, P<0.001). Furthermore, an increase in the number of

eosinophils showing the typical morphological features of apoptosis such as nuclear

coalescense, chromatin condensation and cell shrinkage was found with dextrosimendan

(apoptosis index 0.02 ± 0.01 and 0.98 ± 0.01 in the absence and presence of 40 µM

dextrosimendan, respectively; Figure 3C-D). To further confirm the ability of dextrosimendan

to induce eosinophil apoptosis, DNA breakdown, the typical hallmark of apoptosis was

analyzed. IL-5 inhibited DNA breakdown, and dextrosimendan (40 µM) reversed the IL-5-

afforded inhibition of DNA breakdown and a typical “ladder” pattern was found indicating

the occurrence of apoptotic cell death (Figure 3E). As dextrosimendan was found to be even

more potent in inducing eosinophil apoptosis than levosimendan, dextrosimendan was used in

further studies to evaluate the mechanisms of simendan-induced apoptosis in eosinophils.

Glucocorticoids are known to partially reverse the survival-prolonging action of IL-5 on

eosinophils. However, this effect of glucocorticoids is abolished when IL-5 is used at higher

concentrations (Zhang et al., 2000; 2002; Druilhe et al., 2003; Kankaanranta et al., 2005).

Budesonide (1 µM) partly reversed cytokine-afforded survival in the presence of low (0.1-1

pM) but not in the presence of higher (10-100 pM) concentrations of IL-5 (Figure 4A). To

evaluate whether the effect of dextrosimendan is similar to glucocorticoids, its effects were

studied in the presence of different concentrations of IL-5. Interestingly, increasing

concentrations of IL-5 only partially reversed the effect of low concentration (3 µM) of

dextrosimendan. In contrast, the effect of a higher concentration (10 µM) of dextrosimendan

was not reversed even by high concentrations of IL-5 (Figure 4B).


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Effect of dextrosimendan on Fas-induced eosinophil apoptosis

There are only few compounds that are able to reverse the effect of IL-5 on eosinophil

survival (Kankaanranta et al., 2005). One of those is nitric oxide that has been shown to

reverse the effect of IL-5 by inducing apoptosis (Zhang et al., 2003). However, nitric oxide

can also reverse the apoptosis-inducing effect of Fas in eosinophils (Hebestreit et al., 1998).

This prompted us to evaluate whether dextrosimendan has detrimental effects on Fas-induced

apoptosis. Dextrosimendan (40 µM), further enhanced the apoptosis-inducing effect of Fas-

ligation in human eosinophils as assessed by using the relative DNA fragmentation assay

(apoptotic index 0.71 ± 0.07 and 0.96 ± 0.01 in the absence and presence of dextrosimendan,

n=6, P<0.05) and Annexin-V-binding analysis (59 ± 4% and 96 ± 1% Annexin-V positive

cells in the absence and presence of dextrosimendan, n=6, P<0.001).

Effect of dextrosimendan on spontaneous eosinophil apoptosis

Dextrosimendan enhanced apoptosis of cytokine-deprived eosinophils in a concentration-

dependent manner with an EC50 value of 2.3 ± 0.5 µM (Figure 5). This was confirmed by

morphological analysis of eosinophils cultured for 22 h in the absence and presence of

dextrosimendan (40 µM) (apoptotic indexes 0.21 ± 0.05 and 0.99 ± 0.01, respectively, n=6,

P<0.001). The increased exposure of phosphatidylserine on the outer-leaflet of the cell-

membrane was further confirmed by using Annexin-V binding assay, where the

corresponding percentages of Annexin-V positive cells were 37 ± 5 % and 96 ± 1 % in the

absence and presence of 40 µM dextrosimendan, n=6, P<0.001).

Effect of dextrosimendan on primary eosinophil necrosis

An important feature for a drug possessing anti-eosinophilic activity is that it should not

induce primary necrosis that could lead to the release of eosinophil contents to the


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surrounding tissue. To evaluate this possibility the effects of dextrosimendan on primary

eosinophil necrosis were evaluated by using counterstaining with Annexin-V and propidium

iodide where positive staining with propidium iodide indicates a rupture of the plasma

membrane and the absence of staining with Annexin-V indicates that the cell has not

undergone apoptosis. Thus, cells showing positive staining with propidium iodide (PI) but not

with Annexin-V (Ann-V) can be considered to show the typical feature of primary necrosis,

i.e. the plasma membrane breakdown. In the absence of IL-5, the percentages of PI+/Ann-V-

cells were 4 ± 1% and 1 ± 1% in the absence and presence of 40 µM dextrosimendan,

respectively (n=6, P>0.05). In the presence of IL-5 (10 pM) the corresponding percentages

were 3 ± 1 % and 2 ± 1% (n=6, P>0.05). These results show that dextrosimendan does not

induce primary necrosis in eosinophils.

Effect of caspase inhibition on dextrosimendan-induced apoptosis

A pan-caspase inhibitor, Z-Asp-CH2-DCB (20-200 µM) significantly reversed

dextrosimendan (10 µM)-induced apoptosis in IL-5-treated eosinophils during 40 h

incubation (Table 1).

Role of MAP kinases in dextrosimendan-induced apoptosis in eosinophils

When IL-5-treated eosinophils were incubated at 37 oC in the presence of dextrosimendan (10

µM), a time-dependent increase in the activity of JNK was detected using western blotting

with an anti-pJNK antibody that recognizes the dual phosphorylated (i.e. activated) JNK (Fig

6AB). To evaluate the functional role of JNK activation in dextrosimendan-induced apoptosis

in IL-5-treated cells, a novel cell-permeable inhibitor peptide specific for JNK, L-JNKI1

(Bonny et al., 2001) was employed. L-JNKI1 (10 µM), but not the negative control peptide L-

TAT, almost completely reversed dextrosimendan (10 µM)-induced apoptosis in IL-5-treated


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eosinophils (Fig 6C, 7A-B). To see whether JNK activation is central to the dextrosimendan-

induced apoptosis, the effect of L-JNKI1 (10 µM) on dextrosimendan-induced apoptosis was

analysed by using the morphological analysis and measurement of phosphatidylserine

appearance on the outer cell membrane using Annexin-V binding assay. Interestingly, L-

JNKI1 did not reduce the number of cells showing the typical early signs of apoptosis such as

apoptotic morphology or phosphatidylserine expression on the outer cell membrane. By using

morphological criteria for apoptosis, in dextrosimendan (10 µM) and IL-5 (10 pM) treated

cells the apoptotic indexes were 0.76 ± 0.03 and 0.70 ± 0.03 in the presence of 10 µM L-TAT

and 10 µM L-JNKI1, respectively) after culture for 20 h (Figure 7E-F). Similarly, the

percentage of Annexin-V positive cells were not reduced by L-JNKI1 as compared with cells

treated with L-TAT (76 ± 3% and 71 ± 4 % in the presence of 10 µM L-TAT and 10 µM L-

JNKI1, respectively; Figure 7C-D).

ERK and p38 MAPK have been proposed to be involved in the regulation of

eosinophil apoptosis (Kankaanranta et al., 1999; Hall et al., 2001). Thus, to evaluate the role

of these kinases in dextrosimendan-induced eosinophil apoptosis we used a pharmacological

approach to inhibit the activity of ERKs by using a MEK inhibitor PD098059 and p38 MAPK

by SB203580. However, neither PD098059 (1-10 µM) nor SB203580 (1-10 µM) affected

dextrosimendan (10 µM) -induced apoptosis in IL-5-treated human eosinophils (Table 2).

Effect of KATP channel modulating compounds on eosinophil apoptosis

A report (Yokoshiki et al., 1997) suggested that levosimendan is a KATP channel opener. To

evaluate whether modulation of KATP channel opening state could affect eosinophil apoptosis,

the effects of chemically unrelated KATP channel modulating compounds were studied.

Diazoxide (5-100 µM; a KATP channel opener) only slightly increased the number of apoptotic

eosinophils in the presence of 10 pM IL-5 (apoptotic indexes 0.09 ± 0.01 and 0.17 ± 0.02 in


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the absence and presence of 100 µM diazoxide, respectively, n=5, P<0.01). Similarly, KATP

channel inhibitors glibenclamide (0.03 – 10 µM) and 5-hydroxydecanoate (5-50 µM) were not

able to modify IL-5-afforded eosinophil survival to a significant level. The apoptotic indexes

in IL-5 (10 pM) treated cells were 0.08 ± 0.01 and 0.07 ± 0.01 in the absence and presence of

10 µM glibenclamide, respectively (n=5, P>0.05) and 0.07 ± 0.01 and 0.09 ± 0.01 in the

absence and presence of 50 µM 5-hydroxydecanoic acid (n=5, P<0.01).


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Discussion

In the present study we have shown that the Ca2+ sensitizing and KATP channel

opening simendans reduce pulmonary eosinophilia in mice and induce apoptosis in IL-5-

treated human eosinophils. Furthermore, simendans are able to enhance spontaneous

eosinophil apoptosis without inducing primary necrotic cell death. Their mechanism of action

seems to involve caspase activation as well as JNK-mediated DNA breakdown.

In the treatment of asthma, glucocorticoids reduce the number of eosinophils and

suppress the eosinophilic inflammation. This was evidenced by the reduction of the number of

eosinophils in the BAL fluid of sensitized and challenged mice by budesonide. In addition to

their anti-inflammatory effects, induction of eosinophil apoptosis is currently considered as

one of the mechanisms how glucocorticoids reduce eosinophilic inflammation in asthma

(Druilhe et al., 2003; Walker et al., 2003; Walsh et al., 2003). Clinically, the net effect of

glucocorticoids is a combination of direct anti-eosinophilic effects and suppression of

production of those cytokines that drive the eosinophilic inflammation. Glucocorticoids are

able to enhance spontaneous eosinophil apoptosis at clinically relevant drug concentrations

(Zhang et al., 2000; 2002). In addition, glucocorticoids partly reverse IL-5-afforded survival,

but the effect of steroids falls off as the concentration of IL-5 increases (Zhang et al., 2000;

2002; Druilhe et al., 2003; Kankaanranta et al., 2005). Similarly, in the present study, the

apoptosis-inducing effect of budesonide was abolished by high concentrations of IL-5. In

contrast to glucocorticoids, the effect of dextrosimendan was not reduced by higher

concentrations of IL-5, thus suggesting that the mechanism of action of dextrosimendan is

different from that of glucocorticoids. This suggests that the effect of simendans might be

complementary to that of glucocorticoids. However, there exists the possibility that, in

addition to the induction of eosinophil apoptosis, simendans might also have other anti-

inflammatory effects such as inhibition of synthesis or release of cytokines or chemokines.


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The basic mechanism of action of simendans in the treatment of cardiac failure is that

they bind to the cardiac troponin C to increase its sensitivity to calcium without an increase in

the free intracellular calcium concentration (Haikala H et al., 1995). Although activation of

several, mainly G-protein coupled receptors, is known to lead to an increase in the

intracellular calcium in eosinophils, to our knowledge there is no published literature on the

existence of any of the calcium-binding proteins or troponins in eosinophils. Furthermore, to

our knowledge the only report evaluating the role of free intracellular calcium concentration

([Ca2+]i) in the regulation of eosinophil apoptosis is that by Murray and coworkers (Murray et

al., 2003) showing that a temporary increase in the [Ca2+]i by leukotrienes B4 or D4 is not a

sufficient signal to affect apoptosis in eosinophils. Simendans are known not to increase the

level of [Ca2+]i but to sensitize the effects of cardiac troponin C to calcium. Levosimendan is

approximately 10 times more potent in binding to cardiac troponin C than dextrosimendan

(Sorsa et al., 2004), but is 2-fold less potent in inducing apoptosis in IL-5-treated eosinophils.

This suggests that the apoptosis-inducing mechanism of action of simendans does not depend

on binding to cardiac type troponin C. Whether there exist different types of calcium-binding

proteins in eosinophils that are more sensitive to dextrosimendan than to levosimendan

remains to be elucidated.

Levosimendan has been shown to activate KATP-channels (Yokoshiki et al., 1997).

Glyburide, a blocker of ATP-sensitive K+-channels has been reported to reverse the survival-

prolonging action of IL-5 (Bankers-Fullbright et al., 1998). Interestingly, the effects of

glyburide were not reversed by the KATP-channel opener cromakalim, but cromakalim

potentiated the effects of IL-5 in stressed eosinophils (Bankers-Fullbright et al., 1998). This

prompted us to compare the effects of dextrosimendan to KATP channel modulators. However,

as neither KATP channel opener (diazoxide) nor inhibitors (glibenclamide and 5-


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JPET #124057 19

hydroxydecanoate) produced similar results as dextrosimendan, it is unlikely that the effects

of dextrosimendan are mediated via KATP channel modulation.

Regulation of caspase activity is believed to be central during apoptosis. The presence

of caspases 3,6,7,8 and 9 and their processing during spontaneous or NO-induced apoptosis in

eosinophils has been described (Zangrilli et al., 2000; Dewson et al., 2001; Zhang et al., 2003)

and spontaneous eosinophil death can be blocked by broad specificity caspase inhibitors such

as z-Asp-CH2-DCB or z-VAD-fmk (Dewson et al., 2001; De Souza et al., 2002). However,

the actual caspase pathways mediating the apoptosis in eosinophils remain unknown at the

present (Kankaanranta et al., 2005). The effect of dextrosimendan could be reversed by the

broad specificity caspase inhibitor z-Asp-CH2-DCB, suggesting the involvement of caspase

pathways in its action.

The role of mitogen-activated protein kinases in the regulation of human eosinophil

apoptosis has recently gained attention (Kankaanranta et al., 1999; Hall et al., 2001; Gardai et

al., 2003; Zhang et al., 2003). There exists some controversy whether extracellular regulated

kinase pathway is involved in the survival-prolonging action of cytokines or not

(Kankaanranta et al., 1999; Hall et al., 2001; Kankaanranta et al., 2005), whereas p38 MAPK

seems to be involved in spontaneous eosinophil survival (Kankaanranta et al., 1999). By using

pharmacological inhibitors for MEK and p38 MAPK we were able to exclude ERK and p38

MAPK as targets of dextrosimendan. Recently, JNK has been proposed to be involved in

dexamethasone- (Gardai et al., 2003) and nitric oxide (Zhang et al., 2003)-induced as well as

in spontaneous (Hasala et al., 2007) eosinophil apoptosis. Dextrosimendan induced activation

of JNK as evidenced by Western blot analysis showing an increase in the amount of

phosphorylated JNK. Inhibition of JNK activity by a specific inhibitor L-JNKI1 reversed the

effect of dextrosimendan when apoptosis was measured using the relative DNA fragmentation

assay, suggesting that JNK mediates dextrosimendan-induced apoptosis. However, when the


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effects of L-JNKI1 on dextrosimendan-induced apoptosis were analyzed using morphological

features of apoptosis and the expression of phosphatidylserine on the outer leaflet of the cell

membrane (Annexin-V binding assay), L-JNKI1 was not able to reverse the effect of

dextrosimendan. Taken together, these results suggest that JNK is activated in human

eosinophils in response to dextrosimendan and mediates dextrosimendan-induced DNA

breakdown, but JNK activation is not involved in the early signalling of dextrosimendan-

induced apoptosis. The role of JNK in the regulation of apoptosis in other cell types, mainly

of malignant nature, has been widely studied, and it has been found to have both pro- and

antiapoptotic effects. It may mediate intrinsic apoptosis pathway by inducing cytochrome c

release from mitochondria leading to caspase activation and cell death (Lin and Dibling,

2002; Manning and Davis, 2003). In addition, JNK has been shown to mediate oxidative

stress-induced DNA fragmentation in cardiac smooth muscle cells (Turner et al., 1998). The

exact relationship between JNK activation and DNA fragmentation in eosinophils remains to

be established.

In the present study, dextrosimendan, used at a dose of 1 mg/kg as nasal inhalation,

produced a significant reduction in the number of eosinophils in BAL in sensitized and

challenged mouse. Thus, the concentrations of dextrosimendan administered locally into the

lung reached levels that have remarkable anti-eosinophilic activity. This suggests that the

results obtained are likely to have clinical importance. Interestingly, levosimendan, at doses

similar (0.3-3 mg/kg orally) to those used in the present study, was recently reported to reduce

high-salt diet associated increase in cardiac myocyte apoptosis in hypertensive Dahl/Rapp rats

(Louhelainen et al. 2007). Similarly, levosimendan has been shown to reduce apoptosis in

cardiac myocytes isolated from rat ventricles probably because of the activation of

mitochondrial KATP channels (Maytin and Colucci, 2005). Furthermore, treatment with

levosimendan has been shown to reduce the circulating apoptosis mediators in humans


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JPET #124057 21

(Parissis et al., 2004). This suggests that simendans do not induce apoptosis in all cell types.

However, there reason why simendans induce apoptosis in eosinophils but protect cardiac

myocytes remains unknown at the present.

Taken together, our results suggest that compounds with simendan structure have

remarkable anti-eosinophilic activity and thus are potent candidates for the treatment of

eosinophilic inflammatory conditions in addition to their well-known effect on cardiovascular

performance.


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Acknowledgements

The skilful technical assistance of Mrs. Tanja Kuusela and secretarial help or Mrs. Heli

Maatta are gratefully acknowledged.


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Footnotes

This study was supported by Tampere Tuberculosis Foundation, the Finnish Anti-

Tuberculosis Association Foundation (Finland), the Academy of Finland, the Medical

Research Fund of Tampere University Hospital (Finland), OrionPharma Ltd (Finland) and the

National Technology Agency (TEKES, Finland).

Send reprint requests to:

Hannu Kankaanranta, Department of Respiratory Medicine, Seinajoki Central Hospital,

Hanneksenrinne 7, FIN-60220 Seinajoki, FINLAND. E-mail: [email protected].

Disclosures

RT, EN, MR and HH are current employees of Orion Corporation.


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Legends for Figures

Figure 1. The chemical structures of levo- and dextrosimendans.

Figure 2. Effect of dextrosimendan on the number of BAL eosinophils in ovalbumin-

sensitized and challenged mice. Mice were treated 1 h before and 6 h after each ovalbumin

challenge with dextrosimendan. On 3 consecutive days (days 21-23 from the start of

sensitization), the mice were exposed to aerosols of allergen. Twenty four hours after last

challenge bronchoalveolar lavage (BAL) was collected and the total numbers of eosinophils

were calculated. As a control group, the mice were sensitized with ovalbumin and exposed to

aerosols of 0.9% NaCl. Each data point represents the mean ± SEM, n=17-20. ** indicates

P<0.01 as compared with the control group.

Figure 3. Effect of levosimendan (A) and dextrosimendan on apoptosis in IL-5 (10 pM)

treated human eosinophils during culture for 40 h. Each data point represents the mean ±

SEM of 4 (A) or 6 (B) independent determinations using eosinophils from different donors.

When not visible, error bars are within the symbol size. In (C) is shown the morphology of

eosinophils cultured for 22 h in the presence of IL-5 (10 pM) and in (D) the typical

morphology of apoptotic and late apoptotic eosinophils cultured in the presence of IL-5 (10

pM) and dextrosimendan (40 µM). A representative of 6 similar experiments using

eosinophils from different donors is shown. In (E) is shown the effect of IL-5 (10 pM) and

dextrosimendan (40 µM) on DNA fragmentation in eosinophils cultured for 22 h. A

representative of three similar experiments is shown.

Figure 4. Effect of budesonide (▼; 1 µM; A) and dextrosimendan (■; 3 µM, ▲ ; 10 µM; B)

on apoptosis in eosinophils cultured for 40 h in the presence of different concentrations of IL-


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5. The concentration-curve of IL-5 in the presence of solvent control (0.5% DMSO; B or

0.2% ethanol; A) is indicated by (�). Each data point represents the mean ± SEM of 5

independent determinations using eosinophils from different donors. When not visible, error

bars are within the symbol size. *** indicates P<0.001 as compared with the same IL-5

concentration in the absence of budesonide. In (B) the asterisks have been omitted due to

clarity. All datapoints with either lower (3 µM) or higher (10 µM) dextrosimendan differ

significantly (P<0.001) from the respective control in the absence of dextrosimendan and in

the presence of same concentration of IL-5.

Figure 5. Effect of dextrosimendan on spontaneous apoptosis of cytokine-deprived

eosinophils cultured for 40 h. Each data point represents the mean ± SEM of 6 independent

determinations using eosinophils from different donors.

Figure 6. Effect of dextrosimendan (10 µM) on phosphorylation of c-jun-N-terminal kinase

(JNK) (A). The upper lane shows JNK phosphorylation in the simultaneously prepared

control cells from the same donor treated with solvent control (DMSO 0.5%). A typical

experiment of six similar is shown. B. Density ratio of JNK phosphorylation in

dextrosimendan-treated cells as compared with the simultaneously prepared control cells.

Mean + SEM, n=6. * indicates P<0.05 as compared with 0 min timepoint. C. The effect of L-

JNKI1 (10 µM) and the negative control peptide L-TAT (10 µM) on apoptotic index in

dextrosimendan (10 µM) and IL-5 (10 pM) treated cells. Mean SEM, n=8. *** indicates

P<0.01 as compared with the negative control peptide L-TAT.

Figure 7. Effect of L-JNKI1 (10 µM; B,D,F) on dextrosimendan (10 µM) -induced apoptosis

in IL-5 (10 pM) treated eosinophils as compared with the negative control peptide L-TAT (10


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µM; A,C,E). Representative graphs from relative DNA fragmentation assay of propidium

iodide-stained eosinophils (A,B), Annexin V-FITC (FL-1) and uptake of propidium iodide

(FL-2) (C,D) and morphological analysis of eosinophils (E,F). In C and D figures in top right

hand corner represent total number of early apoptotic eosinophils (annexin V-FITC+ve and PI-

ve) and late apoptotic eosinophils (annexin V-FITC+ve and PI+ve). Representative graphs of

experiments using eosinophils from 4-8 different donors are shown.


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Table 1. The effect of caspase inhibition on dextrosimendan (10 µM)-induced apoptosis in IL-

5-treated eosinophils.

_____________________________________________________________________

Apoptotic index

(mean ± SEM)

_____________________________________________________________________

Solvent control 0.91 ± 0.01

Z-ASP-CH2-DCB

20 µM 0.70 ± 0.02***

200 µM 0.06 ± 0.01***

_____________________________________________________________________

Data is expressed as mean ± SEM, n=6. *** Indicates P<0.001 as compared with the

respective solvent control in the absence of caspase inhibitor. The corresponding apoptotic

index in the presence of IL-5 (10 pM) but in the absence of dextrosimendan was 0.23 ± 0.05.


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Table 2. The effect of MAP kinase inhibitors PD098059 and SB203580 on dextrosimendan

(10 µM) -induced apoptosis in human eosinophils.

_____________________________________________________________________

Apoptotic index

(mean ± SEM)

_____________________________________________________________________

PD098059

0 0.94 ± 0.01

1 µM 0.95 ± 0.01

10 µM 0.96 ± 0.01

SB203580

0 0.91 ± 0.03

1 µM 0.94 ± 0.01

200 µM 0.95 ± 0.02

_____________________________________________________________________

Data is expressed as mean ± SEM, n=5-7. The corresponding apoptotic index in the presence

of IL-5 (10 pM) but in the absence of dextrosimendan was 0.15 ± 0.05.


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Documents

JPET #124057 1 Anti-eosinophilic activity of simendans ...jpet.aspetjournals.org/content/jpet/early/2007/07/09/jpet.107.124057.full.pdfJPET #124057 4 Abstract Simendans are novel agents