ORIGINAL PAPER

Anti-apoptotic therapeutic approaches in liver diseases: do theyreally make sense?

Karen Bannert • Angela Kuhla • Kerstin Abshagen •

Brigitte Vollmar

� Springer Science+Business Media New York 2014

Abstract A variety of data suggesting apoptotic cell

death as a key feature of liver injury stimulated researchers

to investigate the therapeutic potential of anti-apoptotic

strategies in experimental models. However, the overesti-

mated role of apoptotic cell death in liver injury has tem-

pered the clinical translation of the protection afforded by

anti-apoptotic regimes in experimental models. Thus, the

hope for apoptosis modulation as potential treatment

strategy for injured liver in humans could not be confirmed.

Herein, we evaluated the degree of apoptosis in different

hepatic stress models which are relevant for the human

pathophysiology. Using morphological criteria of apopto-

sis, caspase-3 activation as well as TUNEL assay in com-

bination with a positive control of apoptosis in liver injury,

we quantified apoptotic cell death discriminating between

parenchymal and non-parenchymal cells and confirmed

these results by cleaved caspase-3 and PARP-1 protein

expression. Discussing our findings and relating them to

the existing literature on the potential role of apoptotic cell

death, we strongly recommend reconsidering anti-apoptotic

strategies to ameliorate liver injury efficiently.

Keywords Sepsis � Liver cirrhosis � Ischemia

reperfusion � Cholestasis � Caspase-3 � Experimental

Introduction

The liver is a unique organ, that has a potential capacity to

restore the function and mass after part of it has been

removed or damaged. If the remaining liver does not

regenerate adequately, orthotopic liver transplantation rep-

resents the only curative therapeutic option for patients with

advanced liver diseases and hepatic failure. However, poor

long-term graft survival, donor organ shortage and high costs

associated with the procedure call for early treatment of liver

disease. With an improved understanding of the mechanisms

of hepatic cell loss in liver diseases, an era where regulation

of liver cell death is becoming a therapeutic possibility has

been predicted [1, 2]. A variety of data suggesting apoptotic

cell death as a key feature of liver injury stimulated

researchers to investigate the therapeutic potential of anti-

apoptotic strategies in experimental models. However, the

hope for a clinical translation of apoptosis modulation for the

treatment of injured liver in humans could not be confirmed

yet. The overestimated role of apoptotic cell death in liver

injury has tempered the translation of the protection afforded

by anti-apoptotic regimes in experimental models. In this

study, we evaluated the degree of apoptosis in a variety of

hepatic stress models relevant for the human pathophysiol-

ogy in vivo. Apoptotic cell death of parenchymal and non-

parenchymal cells was detected using morphological criteria

of apoptosis, analysis of terminal deoxynucleotidyl trans-

ferase-mediated deoxyuridine triphosphate nick-end label-

ing (TUNEL)-positive cells as well as caspase activation in

combination with a positive control of apoptosis. Addition-

ally, we performed Western blot analysis of cleaved caspase-

3 and the caspase-3 substrate poly-(ADP-ribose) polymer-

ase-1 (PARP-1). We will discuss the findings that emerged

from our study and relate them to the existing literature on

the potential role of apoptotic cell death. Thereby, we will

demonstrate that anti-apoptotic strategies are not sufficient

to ameliorate liver injury efficiently and that misinterpreta-

tion of mechanisms jeopardizes the translation of the find-

ings to the human pathophysiology.

K. Bannert � A. Kuhla � K. Abshagen (&) � B. Vollmar

Institute for Experimental Surgery, Rostock University Medical

School, Schillingallee 69 a, 18057 Rostock, Germany

e-mail: kerstin.abshagen@uni-rostock.de

123

Apoptosis

DOI 10.1007/s10495-014-1004-1

Materials and methods

Animal model

Mice were purchased from Charles River Laboratories

(Sulzfeld, Germany) and were used at 8–10 weeks of age

with a body weight of 23–26 g. Animals were kept on

water and standard laboratory chow ad libitum. The

experiments were conducted in accordance with EU

Directive 2010/63/EU for animal experiments and the

German legislation on protection of animals.

Positive control

As positive control for apoptosis [3], mice were treated

with D-galactosamine (GalN) and Escherichia coli lipo-

polysaccharide (LPS) for 6 h (n = 4).

Negative control

Liver tissue and blood samples from untreated healthy

mice served as negative control for apoptosis (n = 4).

Septic liver

Colon ascendens stent peritonitis (CASP) was used as a

model of polymicrobial abdominal sepsis as it closely

mimics the clinical course of diffuse peritonitis with early

and steadily increasing systemic infection and inflamma-

tion [4]. After opening the abdominal wall of ketamine/

xylazine-anesthetized mice, the colon ascendens was

exteriorized and a 7/0 Ethilon thread was stitched through

the antimesenteric wall approximately 10 mm distal of

ileocecal valve. The prepared catheter was punctured

through the antimesenteric wall directly proximal of the

stitches and fixed by two knots. The inner needle was

removed and cut at the prepared site. To ensure proper

intraluminal position of the stent, stool was milked from

cecum into ascending colon and stent until a small drop of

stool appeared. Mice were killed at 12 h after CASP pro-

cedure (n = 4).

Cholestatic liver

Cholestasis is a common pathological condition that can be

reproduced in rodents by surgical ligation of the common

bile duct [5]. Bile duct ligation (BDL) was performed

under isoflurane anesthesia after midline laparotomy. The

common bile duct was ligated three times with 5-0 silk and

transected between the two most distal ligations. Mice were

killed at 2 days after BDL procedure (n = 4).

Fibrotic liver

Many experimental models of hepatic fibrosis have been

described. None of them completely mimic the spectrum of

liver fibrogenesis seen in humans. Two of the most widely

studied experimental fibrosis models are the surgical liga-

tion of the common bile duct [6] as well as the chronic

intoxication with carbon tetrachloride [7]. Mice were either

killed at 14 days after BDL procedure (see above, n = 4)

or were treated with carbon tetrachloride (2 ml/kg bw ip;

1:4 in corn oil) twice a week for 6 weeks to induce liver

fibrosis (n = 4).

Regenerated liver

Alterations of liver mass are brought about by surgical

removal of tissue to resect tumors and for transplantation

from living donors as well as by functional deficit caused

by chemicals or viruses without loss of mass. Partial hep-

atectomy (pHx) is an excellent experimental model to

study quiescent hepatocytes which become proliferative

and replicate to restore the liver functional capacity as well

as its mass [8]. pHx (70 %) was performed under isoflurane

anesthesia after midline laparotomy. The right upper, the

left upper and the left lower liver lobes were resected by

placing 4–0 silk suture ties most proximally to the origin of

the lobes. Mice were killed at 2 (n = 3) and 8 days (n = 4)

after pHx.

Postischemic liver

Liver injury upon warm hepatic ischemia/reperfusion (I/R)

affects the recovery of patients after major surgery and

bears a risk of poor postoperative outcome. Warm hepatic

I/R can be appropriately mimicked in rodents. Ketamine/

xylazine-anesthetized mice were placed in supine position.

After transverse laparotomy, warm ischemia of the left

liver lobe was induced by transient clamping of the left

hepatic artery and the left portal branch for 60 min, fol-

lowed by removal of the clamp for reperfusion for 60 min

(n = 4).

Liver injury upon cold hepatic I/R is a major obstacle to

liver transplantation. Livers from less-than-optimal donors

develop a profound preservation injury. Organ shortage in

liver transplantation has justified usage of steatotic donor

livers. After transverse laparotomy of ketamine/xylazine-

anesthetized mice (either lean or obese), livers were flushed

via the abdominal aorta with histidine tryptophan keto-

glutarate (HTK) solution, immediately excised thereafter,

weighed and stored in 4 �C HTK solution for 24 h. After

storage, livers were flushed with Ringer’s lactate and then

reperfused for 2 h through the portal vein in a non-recir-

culating fashion with Krebs–Henseleit bicarbonate buffer.

Apoptosis

123

At the end of reperfusion, liver tissue and perfusate sam-

ples were sampled (each n = 4).

Sampling and assay

According to the protocol (see above), mice were anes-

thetized and exsanguinated by puncture of the vena cava

inferior for immediate separation of ethylenediaminete-

traacetic acid plasma. The degree of hepatic disintegration

was assessed by spectrophotometrical determination of

plasma glutamate dehydrogenase (GLDH) and alanine

aminotransferase (ALT) activities using commercially

available reaction kits (Roche Diagnostics, Mannheim,

Germany). Liver tissue was sampled for subsequent Wes-

tern blot protein analysis and immunohistochemistry.

Histology and immunohistochemistry

For histology [hematoxylin & eosin (H&E) staining] and

immunohistochemical analysis of cleaved caspase-3- and

TUNEL-positive cells, liver tissue was fixed in 4 % phos-

phate-buffered formalin for 2–3 days and then embedded in

paraffin. From the paraffin-embedded tissue blocks, 4 lm

sections were put on glass slides and stained with H&E.

Further sections were incubated over night at 4 �C with a

rabbit polyclonal cleaved caspase-3 antibody (1:1,000; Cell

Signaling Technology, Frankfurt, Germany). This antibody

detects endogenous levels of the large fragment (17/19 kDa)

of activated caspase-3 but not full-length caspase-3. A

horseradish peroxidase-conjugated goat anti-rabbit antibody

was used as a secondary antibody (1:2,000; DakoCytomation,

Hamburg, Germany). 3,30-Diaminobenzidine (DakoCyto-

mation) was used as chromogen and the sections were coun-

terstained with hemalaun. For immunohistochemical analysis

of TUNEL-positive liver cells, we used standard protocols as

previously published by our group [9]. Apoptotic cells were

identified by a combination of cleaved caspase-3 and TUNEL

positivity of cells as well as morphological criteria such as cell

shrinkage, chromatin condensation and margination, and

apoptotic bodies [10]. Numbers of apoptotic hepatocytes as

well as non-parenchymal cells were quantified within 15

(TUNEL) to 30 (cleaved caspase-3) consecutive fields (409

objective) and given in % of all visible cells. Images were

acquired with a Color View II FW camera (Color View,

Munich, Germany).

Western blot protein analysis

For Western blot analysis of the protein levels of cleaved

caspase-3/caspase-3 and cleaved PARP-1, liver tissue was

homogenized in lysis buffer (10 mM Tris pH 7.5, 10 mM

NaCl, 0.1 mM EDTA, 0.5 % Triton-X 100, 0.02 % NaN3,

0.2 mM PMSF), incubated for 30 min on ice and centrifuged

for 15 min at 10,0009g. The supernatant was saved as whole

protein fraction. Prior to use, the buffer received a protease

inhibitor cocktail (1:100 v/v; Sigma-Aldrich). Protein con-

centrations were determined using the bicinchoninic acid

protein assay (Sigma-Aldrich) with bovine serum albumin as

standard. 60 lg protein/lane was separated discontinuously

on sodium dodecyl sulfate polyacrylamide gels (14 %) and

transferred to a polyvinyldifluoride membrane (Immobilon-P,

Millipore, Eschborn, Germany). After blockade of non-spe-

cific binding sites, membranes were incubated over night at

4 �C with a rabbit polyclonal anti-cleaved caspase-3 antibody

(Asp 175; 1:1,000; Cell Signaling Technology) or a rabbit

polyclonal anti-PARP-1 antibody (1:1,000; Cell Signaling

Technology), followed by secondary peroxidase-linked goat

anti-rabbit antibody (1:2,000; cleaved caspase-3 and

1:20,000; cleaved PARP-1; Cell Signaling Technology). In

contrast to a cleavage product of PARP-1 at a size of 24 kDa

as shown for several cell lysates [11, 12], the above mentioned

anti-PARP-1 antibody detects a double-band of 35 and

37 kDa as the smallest cleavage products of PARP-1, simi-

larly as described for an alternative anti-PARP-1 antibody

from Abcam (http://www.abcam.com/cleaved-parp-anti

body-ab72805.html). Diverging band sizes could be due to the

type of investigated samples, either whole tissue or cell lysate,

and can even vary between different tissues, probably due to

posttranslational modifications and/or distinct mechanisms in

processing proteins. For example, several fragments of

PARP-1 with different molecular weights have been descri-

bed as signatures of cell-death proteases in case of neurode-

generation [13]. Protein expression was visualized by means

of luminol-enhanced chemiluminescence (Pierce Super Sig-

nal West Dura Extended Duration Substrate, Thermo Scien-

tifc) and digitalized with ChemiDocTM XRS System (Bio-

Rad Laboratories, Munich, Germany). b-actin (mouse

monoclonal anti-b-actin antibody; 1:20,000; Sigma) was used

to verify equal loading of lanes.

Statistics

Data are presented as mean ± standard deviation. After

performing normality test, statistical difference between a

group of liver injury and the negative control group was

determined by Student’s t test. Mann–Whitney rank sum

test was used if criteria for parametric tests were not met.

Data were considered significant when p \ 0.05. Statistical

analysis was performed using the Sigma Plot software

package (Jandel Scientific, San Rafael, CA, USA).

Results

As expected, healthy animals representing the negative

control revealed GLDH activity value below the upper

Apoptosis

123

reference limit of 11.8 U/l in mice [14]. In contrast, ele-

vated plasma GLDH activities in all injury groups studied

reflected the pronounced tissue damage (Table 1). Whereas

septic mice and mice with postischemic livers showed six

to eightfold increased release of GLDH into plasma,

GLDH activities in cholestatic animals after BDL and

animals with liver fibrosis upon CCl4-intoxication were up

to 130- and 300-fold heightened, respectively. In support of

these data, ALT activities were measured as a further

marker of hepatic disintegration. Whereas 8 days regen-

erated livers showed a twofold increase in plasma ALT

(42.7 ± 2.4 U/l), ALT activities in lean livers after cold

ischemia and reperfusion (I/R; 284.0 ± 200.0) and chole-

static livers after BDL (876.8 ± 174.7 U/l) were increased

up to 15- and 60-fold, respectively. In line with this, H&E

histopathology from liver tissue of GalN/LPS-exposed

livers (positive control) exhibited a disruption of liver cell

architecture and microvascular disintegration, accompa-

nied by hepatocellular necrotic cell death (Fig. 1). The

investigated hepatic stress models showed also typical

signs of hepatic injury as inflammatory infiltrate, changes

in the physiological liver structure and necrotic cell death.

Beyond, model-specific changes in liver morphology could

be observed. For example, there were bile infarcts in

cholestatic liver tissue and the accumulation of lipids in

fatty liver after cold I/R (Fig. 1).

Physiologically, apoptosis is almost undetectable in the

liver and only 1–5 apoptotic cells/10,000 cells can be

detected [15]. In line with this, we found at average 3

apoptotic hepatocytes per 10,000 hepatocytes in livers of

healthy animals. However, the number of apoptotic non-

parenchymal liver cells was fivefold higher (Table 1).

Application of GalN/LPS for 6 h (positive control) resulted

in numerous cleaved caspase-3 positive cells, which

showed typical signs of apoptotic cell death (Fig. 2, posi-

tive control). Quantitative analysis resulted in 14 %

apoptotic hepatocytes and 22 % apoptotic non-parenchy-

mal cells (Table 1; Fig. 2). In confirmation with that, we

detected by TUNEL assay comparable, but slightly higher

fractions of apoptotic parenchymal and non-parenchymal

cells with 18 and 30 % (Table 1; Fig. 3).

Compared with healthy animals (negative control), a

significant increase of hepatocellular apoptosis detected by

immunohistochemical analysis of cleaved caspase-3

(Table 1; Fig. 2) could only be observed in fibrotic livers

and in fatty livers after cold I/R. Even so, the number of

apoptotic hepatocytes constituted only *1 % of total

hepatocytes. Extent of hepatocelluar apoptosis of regener-

ated liver tissue as well as of tissue from postischemic lean

livers was negligibly enhanced and never exceeded 0.3 %

of all hepatocytes. Cholestatic liver tissue as well as liver

tissue of septic mice revealed counts of apoptotic hepato-

cytes in the range of physiological values (Table 1; Fig. 2).

Similarly, TUNEL analysis revealed a significant increase

of hepatocellular apoptosis with up to 2 % apoptotic

hepatocytes in septic as well as fibrotic livers and livers

after warm I/R versus negative controls. The percentage of

apoptotic non-parenchymal liver cells is significantly

raised with 4 % in septic livers and 4.5 % in livers after

warm I/R (Table 1; Fig. 3). In addition, compared to

healthy animals, livers after cold I/R exhibited a significant

increase of apoptotic non-parenchymal cells with 7 %. In

contrast to TUNEL analysis, cleaved caspase-3 positivity

accounted for less than 1 % of both parenchymal and non-

parenchymal liver cells except for fibrotic livers upon

CCl4-intoxication and lean livers after warm I/R (Table 1).

Table 1 Plasma concentration of GLDH as well as apoptotic hepatocytes and non-parenchymal liver cells in different hepatic stress models

Hepatic stress model Plasma GLDH (U/l) Apoptotic hepatocytes (%) Apoptotic non-parenchymal liver cells (%)

Cleaved caspase-3 TUNEL Cleaved caspase-3 TUNEL

Positive control (GalN/LPS) 177 ± 104* 13.9 ± 6.53* 18.17 ± 4.82 22.23 ± 8.64* 30.06 ± 13.19*

Negative control 6 ± 2 0.03 ± 0.02 0.26 ± 0.16 0.16 ± 0.03 1.32 ± 0.89

Sepsis 49 ± 2 0.05 ± 0.03 0.61 ± 0.18* 0.18 ± 0.04 4.06 ± 1.03*

Cholestasis 812 ± 132* 0.08 ± 0.07 0.48 ± 0.17 0.14 ± 0.11 1.64 ± 0.40

Fibrosis, BDL 293 ± 56* 1.20 ± 0.35* 2.17 ± 1.10* 0.45 ± 0.17 2.52 ± 0.56

Fibrosis, CCl4 1777 ± 486* 0.76 ± 0.08* 1.01 ± 0.42* 1.45 ± 0.39* 2.04 ± 0.57

Liver regeneration (2 days) 473 ± 247* 0.15 ± 0.08 0.38 ± 0.14 0.25 ± 0.18 1.77 ± 0.51

Liver regeneration (8 days) 39 ± 16 0.13 ± 0.03 0.27 ± 0.20 0.34 ± 0.15 1.20 ± 0.89

Warm I/R of lean liver 40 ± 11 0.27 ± 0.16 1.74 ± 1.32* 2.31 ± 1.56* 4.48 ± 1.05*

Cold I/R of lean liver 41 ± 2 0.26 ± 0.16 0.43 ± 0.33 0.35 ± 0.25 6.95 ± 4.13*

Cold I/R of fatty liver 74 ± 45 0.83 ± 0.38* 1.51 ± 1.05 0.64 ± 0.22 4.44 ± 3.64

Data are given as means ± standard deviations

* p \ 0.05 versus the negative control

Apoptosis

123

Compared to cleaved caspase-3, TUNEL assay resulted

in a slightly higher percentage of apoptotic cells in all of

the investigated hepatic stress models and even in the

negative control which indicates that beside apoptotic also

necrotic cell death was detected in part. Thus, the number

of TUNEL positive cells, e.g. in septic liver, is 12–22-fold

higher versus the number of cleaved caspase-3 positive

cells [hepatocytes: 0.05 ± 0.03 % (cleaved caspase-3) vs.

0.61 ± 0.18 % (TUNEL); non-parenchymal liver cells:

0.18 ± 0.04 % (cleaved caspase-3) vs. 4.06 ± 1.03 %

(TUNEL)]. Nevertheless, the extent of TUNEL-positive

cells still remained markedly less than in GalN/LPS-treated

positive controls (Table 1; Fig. 3).

Western blot protein analysis of cleaved caspase-3

confirmed the results of immunohistochemistry. Liver tis-

sue of mice after GalN/LPS treatment showed a very dis-

tinct staining of both active caspase-3 fragments. In

contrast, liver samples of all injury models studied and, as

expected, the negative control showed only marginal sig-

nals at 17 or 19 kDa (Fig. 4). Moreover, uncleaved cas-

pase-3 was detected as a single band with a molecular

weight of 36 kDa. There were prominent signals in livers

of each stress model as well as the positive and negative

controls without any distinct difference in total caspase-3

expression (Fig. 4). To verify the results of caspase-3

activity, protein expression of caspase-3 substrate PARP-1

was examined. PARP-1 is cleaved by caspase-3 and

therefore considered as a prominent marker of apoptosis.

Liver tissue from healthy mice that served as negative

control exhibited a marginal PARP-1 cleavage indicated by

a single band in Western blots of 35 kDa. In contrast, the

positive controls showed an increased expression of

cleaved PARP-1 characterized by an additional signal of

37 kDa. In liver tissue from all of the hepatic stress models,

a prominent signal of 35 kDa but no or only a weak signal

of 37 kDa was detected. This demonstrates considerably

less caspase-3 activity in the investigated injured livers

compared to the positive controls (Fig. 5).

Discussion

Alternative strategies for the treatment of decompensated

liver diseases are needed to be developed. A thorough

understanding of the underlying mechanisms of liver

damage can offer valuable clues for the development of

Fig. 1 H&E staining. Representative H&E-stained images of the investigated hepatic stress models (magnifications: 910—upper panel, 920—

mid panel, 940—lower panel)

Apoptosis

123

alternative therapeutics. Because experimental data sug-

gested hepatocellular apoptosis as an essential feature in a

wide range of acute and chronic liver diseases, apoptosis-

modulating therapeutics have been within the main targets

of preclinical animal models. However, there is growing

evidence that the impact of apoptosis has been overesti-

mated in a variety of liver diseases [3, 16–24]. Although a

variety of biochemical and immunologic assays have been

developed to detect apoptosis, the most reliable method to

identify apoptotic cell death is morphology [18]. One of the

most exclusive intracellular characteristics of apoptosis

induction is cleavage activation of caspase-3 [25] and

further the cleavage of PARP-1 [26] which is the terminal

pathway independent of the triggering apoptotic signal

[27]. Thus, using morphological criteria as well as caspase-

3 and PARP-1 activation in combination with a reliable

positive control have been suggested to certainly verify

apoptotic cell death in vivo [24]. Herein, we analyzed for

the first time the extent of apoptosis in different hepatic

stress models which are relevant for the human patho-

physiology within a single in vivo study. Whereas multiple

hepatocytes as well as non-parenchymal liver cells were

found to be apoptotic in liver tissue of GalN/LPS mice

(positive control), apoptotic cells were infrequent in livers

of all stress models studied. The number of apoptotic

hepatocytes and non-parenchymal liver cells determined by

immunohistochemical analysis of cleaved caspase-3 never

exceeded 1.2 and 2.3 %, respectively. In addition, analysis

of protein expression of cleaved caspase-3 and PARP-1

verified that apoptotic cell death is rare in livers of all

injured models studied.

The critical role of both forms of cell death, apoptosis or

necrosis, is, however, still controversially discussed in

several forms of liver injury [15, 28–31].

Although TUNEL reliably identifies the internucleoso-

mal DNA cleavage associated with apoptosis, DNA deg-

radation also occurs during necrosis, especially in vivo

because of released nucleases from infiltrating inflamma-

tory cells [22]. Based on this moderate specificity in

exclusively detecting apoptosis, TUNEL data have to be

interpreted carefully. In particular this assay identifies

apoptosis from DNA strand breaks which also appear in

necrotic cells. Nevertheless, we additionally analyzed

apoptotic cell death using TUNEL assay and demonstrated

Fig. 2 Immunohistochemistry of cleaved caspase-3. Representative

images of cleaved caspase-3 immunohistochemistry of liver tissue of

different liver disease models (each n = 4). For details, please see

text. Bars 100 lm (lower magnification) and 10 lm (higher magni-

fication, see insets)

Apoptosis

123

that the extent of apoptotic cell death was indeed higher as

analyzed by cleaved caspase-3, but was still moderate.

Thus, TUNEL assay assessed a minor percentage of

necrotic cells in all models studied. However, the high

levels of ALT and GLDH display a much higher extent of

necrotic tissue damage than identified by TUNEL assay.

Fig. 3 TUNEL assay. Representative images of TUNEL immunohistochemistry of liver tissue of different liver disease models (each n = 4).

For details, please see text. Bars 100 lm (lower magnification) and 10 lm (higher magnification, see insets)

Fig. 4 Western blot protein analysis of cleaved caspase-3. Immuno-

blots of caspase-3 and cleaved caspase-3 of liver tissue of different

liver disease models (each n C 3). Liver tissue of the positive control

(plus) showed a very distinct staining, whereas livers of the healthy

negative control (minus) as well as the diseased animals exhibited

only marginal signals at 17 and 19 kDa. Expression levels of total

caspase-3 (36 kDa) were comparable in hepatic stress models as well

as the positive and negative controls. b-actin was used to verify equal

loading of lanes

Apoptosis

123

Therefore, TUNEL assay is indeed unspecific and detects

rather apoptosis than necrosis.

Jaeschke and co-workers [16, 18] have already con-

vincingly demonstrated that necrosis instead of apoptosis is

the principal mechanism of cell death of both hepatocytes

and non-parenchymal cells after ischemia and reperfusion

of the liver. Only less than 2 % of hepatocytes as well as

sinusoidal endothelial cells were observed to be apoptotic.

Herein, we confirmed that apoptotic cell death plays only a

minor role in the development of hepatic I/R injury after

both warm and cold ischemia. However, numerous studies

have suggested that the non-parenchymal liver cells (i.e.,

sinusoidal endothelial and Kupffer cells) are major targets

of cold ischemic injury and that these cells undergo

apoptosis [32–34]. These findings could not be confirmed

by the present work, which goes along with data by Huet

et al. [20] showing that death of sinusoidal endothelial cells

occurs by necrosis during the early phase of warm

reperfusion.

Ursodeoxycholic acid (UDCA) is currently considered

the first choice for many forms of cholestatic hepatopathies

[35]. Although used in an empirical manner, it is the only

disease-modifying drug therapy with evidence of efficacy

[36]. Many mechanisms and sites of action have been pro-

posed for UDCA, but definitive data are still missing

regarding the key points of its efficacy in order to achieve a

sustained clinical effect [35]. Among the suggested mech-

anisms of action, protection of hepatocytes against bile acid-

induced apoptosis has been suggested [35, 37, 38]. It is

thought that UDCA induces anti-apoptotic signals via

stimulation of the intracellular mitogen-activated protein

kinase (MAPK) as well as phosphatidylinositol 3-kinase

(PI3K) signaling pathways and inhibits the mitochondrial

membrane permeability [39, 40]. However, the relevance of

these findings for human cholestasis is unclear because

effects of UDCA in rodents were only partially reproduced

in humans [41, 42]. The involvement of the MAPK and PI3K

pathways in the anti-apoptotic protection of UDCA has been

demonstrated in vitro during bile acid induced apoptosis of

rat hepatocytes [43, 44]. Similarly, reduced alterations in

mitochondrial function by taurine-conjugated UDCA have

been shown in bile acid caused mitochondrial permeability

transition of mitochondria isolated from rat liver [45]. For

stimulation of apoptosis in cultured rodent hepatoctyes, bile

acid concentrations of 50 lM or above are needed. How-

ever, maximal concentration in serum of bile duct ligated

mice are 1,000-fold less and are not sufficient to stimulate

hepatocyte apoptosis in vivo [46]. In line with this, we

observed herein a negligible number of apoptotic cells in

cholestatic animals. Woolbright and Jaeschke [24] sug-

gested that hepatotoxic bile acid concentrations initiate an

inflammatory response and cell death by neutrophils through

oxidative stress. Indeed, UDCA has direct antioxidant

properties [40]. In a model of inflammation induced chole-

static liver injury, feeding with a homologue of UDCA,

almost completely eliminated portal neutrophil infiltration

as well as attenuated the inflammatory response and oxida-

tive stress [47]. A close correlation between the improve-

ment in the imbalance of lymphocyte subsets after UDCA

therapy of patients with primary biliary cirrhosis and the

clinical status suggests that an immunological process plays

a role in the effectiveness of therapy [48]. Taken together,

the hepatoprotective action of UCDA in cholestatic liver is

unlikely attributed to anti-apoptotic mechanisms. Increasing

information on the cause-and-effect relationship of UDCA

treatment will represent a major step towards the develop-

ment of novel, more effective therapeutic strategies against

cholestatic syndromes.

Fig. 5 Western blot protein analysis of cleaved PARP-1. Immuno-

blots of PARP-1 of liver tissue of different liver disease models

(n = 1–4). Liver tissue from healthy mice that served as negative

control (minus) exhibited a marginal PARP-1-cleavage indicated by a

single band of 35 kDa. In contrast, the positive control (plus) showed

an increased expression of cleaved PARP-1 characterized by an

additional signal of 37 kDa. In liver tissue of all hepatic stress

models, a prominent signal of 35 kDa but no or only a weak signal of

37 kDa was detected. b-actin was used to verify equal loading of

lanes

Apoptosis

123

Chronic forms of extrahepatic as well as of intrahepatic

cholestasis can culminate in liver fibrosis, which is a basic

step in the progression to cirrhosis. In both extrahepatic

(caused by biliary obstruction) and intrahepatic (caused by

drug-induced liver injury) models we failed to detect

notable extent of apoptotic cell death. Fibrotic livers after

bile duct ligation revealed the highest numbers of apoptotic

hepatocytes in all models studied, but about 1 % apoptotic

cells are most probably of minor pathophysiological rele-

vance and therefore an inadequate therapeutic target site. In

contrast, induction of apoptosis of hepatic stellate cells

might suppress and even reverse liver fibrosis, since acti-

vated stellate cells are the source of collagen formation

[22] and hepatic stellate cell apoptosis is a vital mechanism

that contributes to recovery from hepatic fibrosis [49].

Similarly, there is no evidence to target apoptotic cell

death as therapeutic strategy during liver regeneration after

pHx. Although stated that after massive liver resection,

activation of apoptosis rather than mitogenic pathways

results in liver failure [50], the respective studies did not

proof the presence of this kind of cell death [51, 52].

Abshagen et al. [53] observed a maximal but only slight

increase of apoptotic cell rate at 5 days after pHx when the

number of proliferating cells already decreased. In contrast,

Sakamoto et al. [54] reported on a wave of apoptosis

between 60 and 96 h after pHx in mice which was directly

proportional to the hepatocyte BrdU labeling. However, a

range of 0–5 (median: 2.0) apoptotic hepatocytes per 20

high power fields at 96 h after pHx compared to 0–2

(median: 0.0) apoptotic cells in non-hepatectomized ani-

mals [54] seems to be of minor relevance, but corresponds

well to the about fourfold increase observed in our study.

Rapid proliferation tends to produce functionally insuffi-

cient cells and architecture [55]. Elimination of cells via

apoptosis is a common event in processes involving organ

growth [56]. Anti-apoptotic strategies do not seem to be

advisable because balance between apoptosis and hepato-

cyte survival is critical for appropriate liver regeneration

and remodeling [57, 58].

Hepatic dysfunction is one of the characteristics of criti-

cally ill, in particular septic patients and is associated with

worse outcome. Dysregulation of cytokines such as tumor

necrosis (TNF) mainly produced by macrophages including

Kupffer cells has been found to correlate with severity of liver

failure in humans. To simulate systemic inflammatory

response syndrome, application of bacterial cell wall consti-

tutes such as LPS is commonly used in animal models. As

rodents are known to be more than 1000-fold less sensitive

towards LPS than humans, they are sensitized by pretreatment

with the amino sugar GalN [59]. LPS-challenged mice pre-

treated with GalN exhibit extensive hepatocellular apoptosis

mainly dependent on TNF-a, which is secreted by LPS

stimulated Kupffer cells [59, 60]. Therefore, TNF-dependent

apoptotic cell death has been suggested as a common patho-

logical process during liver damage associated with bacteria

[59, 61]. GalN is exclusively metabolized in hepatocytes

leading to severe transcription and translation arrest as early as

30 min after injection, which sensitizes the liver towards

TNF-a [59]. TNF-a activates the transcription factor nuclear

factor jB (NFjB), which translocates into the nucleus within

30 min to 4.5 h after GalN/LPS treatment. The shuttle back of

NFjB to the cytoplasma is disturbed upon GalN application

and approximately 30 % of NFjB remains in the nuclear

fraction [60]. Application of TNF-a or LPS alone without

transcriptional blockade is insufficient to induce hepatocyte

apoptosis [2, 59, 62, 63]. Herein, we confirm in a model of

polymicrobial abdominal sepsis that apoptotic liver cell death

is of minor relevance in systemic inflammatory response

syndrome.

Conclusion

In none of the studied hepatic stress models we observed a

rate of apoptotic cell death with distinct pathophysiological

relevance. These findings strongly argue against apoptotic

cell death as a therapeutic target in these kinds of liver

diseases. Because caspases are major executors of the

apoptotic program in neutrophils, inhibition of caspase

activation can hinder neutrophil apoptosis and so may

prolong or even worsen their inflammatory response [64].

Moreover, decreased apoptosis can be compensated by

other forms of cell death, which in the worst case would be

more deleterious to the organ [22, 65]. Beside this, con-

cerns about their potential carcinogenicity limit the thera-

peutic application of anti-apoptotic approaches.

Taken together, our data challenge the notion of apop-

totic cell death as a key feature of liver injury, and call into

question the preclinical basis for clinical studies exploring

therapeutic potential of anti-apoptotic strategies.

Acknowledgments This work was supported in part by the Deut-

sche Forschungsgemeinschaft, Bonn-Bad Godesberg, Germany (Ei

768/1-2; AB 453/1-1). The authors kindly thank Berit Blendow,

Dorothea Frenz, Eva Lorbeer-Rehfeldt and Maren Nerowski (Institute

for Experimental Surgery, University of Rostock) for excellent

technical assistance.

Conflict of interest All authors declare that they have no conflicts

of interest.

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