ORIGINAL PAPER

Chronic protection against ischemia and reperfusion-inducedendothelial dysfunction during therapy with different organicnitrates

Monica Lisi • Matthias Oelze • Saverio Dragoni • Andrew Liuni •

Sebastian Steven • Mary-Clare Luca • Dirk Stalleicken • Thomas Munzel •

Franco Laghi-Pasini • Andreas Daiber • John D. Parker • Tommaso Gori

Received: 18 July 2011 / Accepted: 16 January 2012 / Published online: 2 February 2012

� Springer-Verlag 2012

Abstract

Introduction Ischemic and pharmacologic precondition-

ing have great clinical potential, but it remains unclear

whether their effects can be maintained over time during

repeated exposure.We have previously demonstrated that

the acute protective effect of nitroglycerin (GTN) is

attenuated during repeated daily administration. Pentaery-

thrityl tetranitrate (PETN) is an organic nitrate with

different hemodynamic and biochemical properties. The

purpose of the current experiment was to study the

preconditioning-like effects of PETN and GTN during

repeated daily exposure.

Methods and results In a randomized, investigator-blind

parallel trial, 30 healthy (age 25–32) volunteers were ran-

domized to receive (1) transdermal GTN (0.6 mg/h)

administered for 2 h a day for 6 days; (2) oral PETN

(80 mg) once a day for 6 days; or (3) no therapy. One week

later, endothelium-dependent flow-mediated dilation was

assessed before and after exposure to ischemia and reper-

fusion (IR). IR caused a significant blunting of the endo-

thelium-dependent relaxation in the control group (FMD

before IR: 5.8 ± 2.1%; after IR 1.0 ± 2.1%; P \ 0.01).

Daily, 2-h exposure to GTN partially prevented IR-induced

endothelial dysfunction (FMD before IR: 7.7 ± 2.4%; after

IR 4.3 ± 3.0%; P \ 0.01 compared to before IR). In

contrast, daily PETN administration afforded greater pro-

tection from IR-induced endothelial injury (FMD before

IR: 7.9 ± 1.7%; after IR 6.4 ± 5.3%, P = ns; P \ 0.05

ANOVA across groups). In vitro, incubation of human

endothelial cells with GTN (but not PETN) was associated

with inhibition (P \ 0.01) of aldehyde dehydrogenase, an

enzyme that is important for both nitrate biotransformation

and ischemic preconditioning.

Discussion We previously showed that upon repeated

administration, the preconditioning-like effects of GTN are

attenuated. The present data demonstrate a gradient in the

extent of protection afforded by the two nitrates, suggesting

that PETN-induced preconditioning is maintained after

prolonged administration in a human in vivo model of

endothelial dysfunction induced by ischemia. Using iso-

lated human endothelial cells, we propose a mechanistic

explanation for this observation based on differential

effects of GTN versus PETN on the activity of mitochon-

drial aldehyde dehydrogenase.

Keywords Nitroglycerin � Ischemia and reperfusion �Ischemic preconditioning � Endothelial function �Pentaerythrityl tetranitrate

Abbreviations

GTN Nitroglycerin

IR Ischemia–reperfusion

PETN Pentaerythrityl tetranitrate

M. Lisi and M. Oelze equally contributed to this paper.

M. Lisi � S. Dragoni � F. Laghi-Pasini

Department of Clinical Medicine and Immunological Sciences,

University of Siena, Siena, Italy

M. Oelze � S. Steven � T. Munzel � A. Daiber � T. Gori (&)

2. Medizinische Klinik, University Medical Center Mainz,

55131 Mainz, Germany

e-mail: Tommaso.gori@unimedizin-mainz.de

A. Liuni � M.-C. Luca � J. D. Parker

Department of Cardiology, Mont Sinai and University Health

Network Hospitals, Toronto, Canada

D. Stalleicken

Actavis Deutschland GmbH, Langenfeld, Germany

123

Clin Res Cardiol (2012) 101:453–459

DOI 10.1007/s00392-012-0412-x

Introduction

Organic nitrates have long been used for their vasodilator

and antianginal effects; their clinical utility is however

limited by the development of tolerance, i.e. the loss of

hemodynamic effects that is observed upon prolonged

administration (reviewed in [1]). Beyond their hemody-

namic properties, it is now recognized that organic nitrates

also have potent protective effects that are similar to those

of ischemic preconditioning. Single, short-term exposure to

nitroglycerin (GTN) has been associated with reduced

evidence of ischemia in vitro [2], in animals [3], in patients

during both exercise stress test and angioplasty [4, 5] as

well as with a reduced endothelial dysfunction after

ischemia and reperfusion (IR) injury [6, 7].

Similar to hemodynamic tolerance, also the precondi-

tioning effects of GTN appear to be attenuated during

repeated, 2 h-daily administration [8, 9]. This phenome-

non, whose mechanism(s) remain unknown, appear to

depend at least in part (like hemodynamic tolerance) on

oxidative stress, as administration of the antioxidant vita-

min C is able to recapture the preconditioning effect of

GTN [8]. Importantly, the development of such ‘‘toler-

ance’’ to the preconditioning effects of GTN would limit

any therapeutic plan designed to exploit a preconditioning

effect of daily exposure to this organic nitrate.

A number of studies have shown that important differ-

ences exist across different nitrates in terms of their sus-

ceptibility to hemodynamic tolerance [10]. For instance, the

vasodilatory effects of pentaerythrityl tetranitrate (PETN)

are preserved upon chronic administration, a phenomenon

that might depend on intrinsic antioxidant properties of this

molecule and on the induction of heme-oxygenase-1, an

enzyme involved in both antioxidant protection and ische-

mic preconditioning [11]. While it is known that PETN, like

GTN, possesses acute preconditioning properties [7], the

present study was designed to test, in humans, whether

prolonged PETN administration is associated with precon-

ditioning protection, and whether there is a gradient across

different drugs in the extent of this protection. In human

isolated endothelial cells, we studied the role of the aldehyde

dehydrogenase, a mitochondrial enzyme that appears to be

involved in both the bioactivation of organic nitrates [12]

and in the protection conferred by ischemic and pharma-

cologic preconditioning [13].

Methods

The Ethics Committee of the University Hospitals of Siena,

Italy and Mainz, Germany, approved the protocols. Written

informed consent was obtained from all participants.

In an investigator-blinded, parallel trial, 30 healthy (age

range 25–32 years) volunteers were randomized to one of

three groups: (1) transdermal GTN (0.6 mg/h) adminis-

tered for 2 h a day for 6 days; (2) oral PETN 80 mg o.d. for

6 days; (3) no therapy. Seven days after the randomization

(24 h after the last administration of the study medica-

tions), all subjects underwent measurement of endothe-

lium-dependent flow-mediated dilation (FMD) before and

after induction of local IR (15 min of ischemia followed by

15 min of reperfusion) as previously described [7]. To

improve the reproducibility and repeatability of the method,

a deflation pillow was used to immobilize the patients’

forearm, a stereotactic clamp with micrometric screws was

used to support the ultrasound probe, and arterial diameter

analysis was performed in an investigator-blinded fashion

using dedicated software (SpLiNeS, Siena, Italy [14]). This

human in vivo forearm model is a well-consolidated method

to specifically study IR-induced endothelial dysfunction

while leaving smooth muscle responses unaltered; the

impairment in endothelial responsiveness observed in these

studies can be prevented by ischemic and pharmacologic

(including GTN-mediated) preconditioning [7].

Cell culture and treatment

Human endothelial cells EA.hy 926 cells were a kind gift

of C. J. Edgell (University of North Carolina at Chapel

Hill, USA) [15] and were grown in Dulbecco’s modified

Eagle’s medium (Sigma) with 10% fetal calf serum, 2 mM

L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin,

100 lg/ml streptomycin, and 19 HAT (hypoxanthine,

amethopterin/methotrexate, thymine). For experiments, the

cells were seeded in six-well plates at a density of

2 9 105 cells/well and grown to 60–70% confluence until

the experiment was started. Cells were kept in the medium

and exposed to GTN or PETN (5 lM for 24 h) or subjected

to no therapy for four consecutive days. Twenty-four hours

after the last exposure to the nitrates, the medium was

removed and cells were scraped in some plates in 0.4 ml

Laemmli buffer and frozen at -80�C until Western blot

analysis. Cells in other plates were used for determination

of ALDH-2 activity.

Western blot analysis for ALDH-2 expression

Western blot analysis was performed as previously

described [16]. Laemmli cell samples were subjected to

Western blot analysis. Proteins were separated by SDS-

PAGE and blotted onto nitrocellulose membranes. After

blocking, immunoblotting was performed with antibodies

against b-actin (42 kDa) (1:2,500, Sigma-Aldrich) as

controls for loading and transfer and polyclonal rabbit

ALDH-2 (1:2,000, kindly provided by K K. Ho and H.

454 Clin Res Cardiol (2012) 101:453–459

123

Weiner, Purdue University, West Lafayette, USA). Detec-

tion was performed by enhanced chemiluminescence with

peroxidase conjugated anti-rabbit (1:10,000, Vector Lab.,

Burlingame, CA) secondary antibody. The antibody-specific

bands were quantified by densitometry as described.

HPLC analysis for ALDH-2 activity

Cells were incubated for 45 min with Monal 62 (50 lM) in

phosphate-buffered saline (PBS) and 50 ll of the supernatant

was used for HPLC analysis. The oxidation of 6-methoxy-2-

naphthylaldehyde (Monal 62) to the fluorescent naphthoic

acid product [17] was traced by HPLC analysis. The system

consisted of a control unit, two pumps, mixer, detectors,

column oven, degasser, and an autosampler (AS-2057 plus)

from Jasco (Groß-Umstadt, Germany) and a C18-Nucleosil

100-3 (125 9 4) column from Macherey & Nagel (Duren,

Germany). A high pressure gradient was employed with

acetonitrile/water (90/10 v/v%) and 25 mM citrate buffer, pH

2.2, as mobile phases with the following percentages of the

organic solvent: 0 min, 40%; 8 min, 60%; 8–10 min, 100%;

11 min, 40%. The flow was 1 ml/min and naphthoic acid was

detected by fluorescence (Ex. 310 nm/Em. 360 nm).

Statistical analysis

Results are expressed as mean ± SD. Within- and

between-group differences for all variables were evaluated

with two- or one-way ANOVA for separate or repeated

measures, as appropriate. The Bonferroni correction was

used for post hoc comparisons. P \ 0.05 was set as the

threshold for significance.

Results

Hemodynamic data

Data are presented in Table 1 and Fig. 1.

Before IR, there was no difference across groups in

heart rate, blood pressure, blood flow, resting radial artery

diameter or FMD (P = ns for all).

IR caused a significant blunting of the endothelial

responses in the control group (Fig. 1, P \ 0.01 versus

before IR).This effect was partially prevented in the group

that received treatment with GTN (Fig. 1, P = ns com-

pared to control group on ANOVA). Confirming our pre-

vious observation that the preconditioning-mimetic

properties of GTN partially wane upon repeated adminis-

tration [8], FMD after IR was significantly blunted in the

GTN group (P \ 0.01 compared to before IR, within

group). In contrast, FMD was not modified by IR in the

PETN group (P = ns compared to before IR). Differences

across groups were confirmed by ANOVA analysis

(P \ 0.01).

Table 1 The effect of IR and nitrate therapy on hemodynamic parameters and IR-induced endothelial dysfunction

Blood pressure Heart rate Radial artery diameter (mm)

SBP DBP Before IR After IR

Basal DFMD Basal DFMD

No therapy 115 ± 3 73 ± 3 78 ± 2 2.06 ± 0.11 0.13 ± 0.01 2.12 ± 0.11 0.02 ± 0.02*

GTN 116 ± 3 68 ± 2 70 ± 3 2.13 ± 0.12 0.16 ± 0.02 2.23 ± 0.13 0.10 ± 0.02*

PETN 110 ± 3 70 ± 3 68 ± 4 2.24 ± 0.11 0.18 ± 0.02 2.27 ± 0.12 0.14 ± 0.04

Hemodynamic parameters were similar across before IR and were not affected by IR. FMD was significantly blunted by IR in the placebo and

(although to a lesser extent) the GTN group

SBP systolic blood pressure in mmHg, DBP diastolic blood pressure, DFMD diameter increase during reactive hyperemia

* P \ 0.01 (ANOVA) compared to corresponding DFMD before IR

0

1

2

3

4

5

6

7

8

FMD9%

Before IR After IR

PETNGTNCtr

P<0.01

P<0.01

P= ns

P<0.05

P= ns

Fig. 1 FMD (flow-mediated dilation) before (left) and after (right)ischemia and reperfusion (IR). FMD was significantly blunted in the

control group and the GTN group, but not in the PETN group. In

contrast, IR had no effect of FMD in PETN-treated subjects. n = 11

for GTN and PETN, n = 8 for placebo

Clin Res Cardiol (2012) 101:453–459 455

123

ALDH-2 protein expression and activity data

Data are presented in Fig. 2. Incubation with repeated

dosages of GTN (5 lM) was associated with a decrease in

the expression of the protective enzyme ALDH-2

(P \ 0.05 vs. control). In contrast, repeated PETN incu-

bations (each 5 lM) were not associated with a loss of the

expression of ALDH-2 (P = NS vs. control; P \ 0.05 vs.

GTN). These observations were mirrored by the ALDH-2

activity, which was markedly decreased in response to

chronic GTN treatment and only marginally altered by

PETN incubations.

Discussion

A number of studies have investigated the existence and

mechanism(s) of preconditioning protection induced by a

variety of pharmacological stimuli (including a single

exposure to organic nitrates). Both the extent of protection

and the molecular mechanisms of pharmacologic precon-

ditioning appear to be similar to those induced by classical

ischemic preconditioning [2]; as such, pharmacologic

preconditioning might represent a promising approach to

long-term protection in patients at risk. Similar to the

endothelial effect of other drugs [18], however, the clinical

relevance of this strategy is dependent on whether the

protection induced can be maintained over prolonged or

repeated exposure to the preconditioning stimulus. Of note,

few studies have investigated whether such ‘‘chronic’’

preconditioning actually exists. Emphasizing the existence

of differences between ‘‘short-term’’ and ‘‘chronic’’ pre-

conditioning, a recent animal model found that, despite

similar protection from IR, the pattern of genomic activa-

tion induced by a single preconditioning stimulus is pro-

foundly different from that induced by the repetition of the

same stimulus [19, 20]. Thus, while this protective phe-

nomenon may in certain cases be preserved, it should not

be assumed that ‘‘short-term’’ preconditioning can in all

cases be translated into long-lasting (clinically more rele-

vant) protection.

In the present study, repeated administration of PETN

resulted in continued protection against IR-induced endo-

thelial dysfunction. In line with our previous findings [8],

the protection induced by repeated daily GTN administra-

tion was attenuated, with an FMD post-IR that was inter-

mediate between that in the placebo and the PETN groups,

a situation that is analogous to the hemodynamic effects

of the two drugs during prolonged continuous therapy.

The present data also suggest a possible mechanism for

this observation. PETN is an organic nitrate that, like

GTN, undergoes biotransformation catalyzed by the

ALDH-2

0

20

40

60

80

100

120

Ctr GTN PETN

% o

f D

MS

O *

#

AL

DH

-2 a

ctiv

ity

by M

onal

oxid

atio

n [µ

M]

Ctr GTN PETN0

1

2

3

4

P<0.05

P<0.05A B

Fig. 2 Effect of repeated GTN (49 GTN) or PETN (49 PETN)

administration on the protein expression (a) and enzymatic activity

(b) of ALDH-2 as assessed by Western blot and HPLC analysis.

Expression is presented as a percentage of the corresponding vehicle

and original blots are shown for two ALDH-2 and loading control

(b-actin) samples in each treatment group below the columns.

Activity is presented as absolute conversion of a naphthoic aldehyde

(Monal 62) to its fluorescent naphthoic acid product. Representative

HPLC chromatograms are shown for each treatment group below the

columns. All mean values ± SEM are based on data from 13–15

(protein expression) and 4–8 (enzymatic activity) independent cell

culture samples

456 Clin Res Cardiol (2012) 101:453–459

123

mitochondrial ALDH-2 to release its active metabolite,

nitric oxide or a nitric oxide adjunct [21]. This drug is used

in the therapy of stable angina but a benefit has also been

shown in heart failure [22]. Due to pharmacodynamic

differences and to intrinsic antioxidant properties, PETN

appears to be devoid of hemodynamic tolerance (reviewed

in [23, 24]). While evidence of GTN-induced (but not

PETN-induced [21]) oxidative inhibition and reduced

expression of the ALDH-2 in whole vessels and isolated

smooth muscle cells has been reported previously [25, 26],

our findings confirm these findings in endothelial cells, and

provide an interesting link with the preconditioning-like

effects of the two drugs. The ALDH-2 inhibition observed

here in vitro fits well with the loss of GTN’s precondi-

tioning-like effects in vivo reported in our previous paper

[8]; the evidence that administration of an antioxidant is

able to recapture GTN-induced preconditioning-like pro-

tection suggests that oxidative inhibition of this enzyme (as

in the case of hemodynamic tolerance) might be respon-

sible for this observation. Importantly, animal studies have

recently shown that both physical and pharmacological

preconditioning stimuli converge in the activation of the

ALDH-2, and that pharmacological inhibition of ALDH-2

inhibits effective preconditioning [13, 27]. Compatible

with this concept, the inhibition of this enzyme observed in

endothelial cells in our in vitro studies might result in a

lesser endothelial protection (i.e., in an increased suscep-

tibility to IR-induced endothelial dysfunction) compared to

PETN. Notably, we previously showed that the mito-

chondrial production of reactive oxygen species plays a

key role in the GTN-induced preconditioning [6]. The

present data are compatible with the hypothesis that, upon

repeated administration, oxidative modifications in the

ALDH-2 might cause loss of function of this enzyme,

leading to reduced GTN biotransformation and inhibition

of the end-effector of both ischemic and pharmacologic

preconditioning.

Some limitations need to be acknowledged: first, healthy

subjects were enrolled, and the peripheral circulation was

tested in this experimental model. Although abnormalities

in FMD have been shown in many conditions [28–33], like

for any model, the translation of the present data to the

clinical setting (patients with coronary artery disease,

coronary or cerebral circulation) needs to be further

investigated. Similarly, the data from isolated endothelial

cells should not be automatically transferred to the clinical/

human in vivo setting, and additional mechanisms could

explain our observations, even though we previously

showed human in vivo ALDH-2 inhibition in response to

GTN [34]. As well, the two drugs have different pharma-

codynamic properties, and PETN has a longer half-life than

GTN (8–10 h vs. 20–30 min) [35]. Although no hemody-

namic effect was present at 24 h, a human in vivo setting

does not allow excluding that the effect of PETN observed

here might be at least in part mediated by anti-ischemic

properties of hemodynamically inactive PETN metabolites

and that they might be independent of traditional precon-

ditioning mechanisms. Whatever the mechanism, however,

the preservation of endothelial function despite IR deserves

investigation in clinical settings.

In sum, while the present data provide further mecha-

nistic evidence for the preconditioning effect of organic

nitrates, they also emphasize the analogies between the

mechanisms leading to ‘‘hemodynamic’’ nitrate tolerance

and nitrate-induced preconditioning. At the level of whole

vessels, previous studies emphasized the possible role of an

oxidative ALDH-2 inhibition in determining tolerance to

the hemodynamic effects of GTN (but not PETN) [21, 25].

We show that inhibition of the ALDH-2 by GTN also

occurs in isolated endothelial cells and propose that this

phenomenon might alter the susceptibility to IR-induced

endothelial dysfunction in vivo in humans. Thus, the

functional status of the ALDH-2 might be important not

only for the metabolism and bioactivation of organic

nitrates, but also for tissue recovery in the setting of

ischemia. This evidence should be seen in light of pre-

liminary studies reporting an increased incidence of car-

diovascular events in patients undergoing nitrate therapy

[36], and emphasizes the need of a prospective, random-

ized trial on the clinical long-term impact of these drugs.

Importantly, preconditioning might have clinical implica-

tions [37], but our data emphasize the complexity of this

phenomenon and support the concept that the protection

observed in response to a single exposure to a precondi-

tioning stimulus should not be assumed to imply that the

same degree of protection would be observed in the chronic

setting. Future studies testing the applicability of precon-

ditioning as a clinical tool should also test whether this

effect is maintained over a prolonged time.

Acknowledgments We thank Angelica Karpi and Nicole Papa-

ioannou for expert technical assistance. John Parker is the recipient of

a Grant from the Canadian Institute for Health Research. The study

was funded by an unrestricted grant from Actavis Deutschland

GmbH. DS is an employee of Actavis Deutschland; TG, AD and MO

have received speaker’s bureaus from Actavis Deutschland.

References

1. Munzel T, Daiber A, Gori T (2011) Nitrate therapy: new aspects

concerning molecular action and tolerance. Circulation 123(19):

2132–2144. doi:10.1161/CIRCULATIONAHA.110.981407

2. Bolli R, Dawn B, Tang XL, Qiu Y, Ping P, Xuan YT, Jones WK,

Takano H, Guo Y, Zhang J (1998) The nitric oxide hypothesis of

late preconditioning. Basic Res Cardiol 93(5):325–338

3. Szilvassy Z, Ferdinandy P, Nagy I, Jakab I, Koltai M (1997) The

effect of continuous versus intermittent treatment with transder-

mal nitroglycerin on pacing-induced preconditioning in conscious

Clin Res Cardiol (2012) 101:453–459 457

123

rabbits. Br J Pharmacol 121(3):491–496. doi:10.1038/sj.bjp.

0701163

4. Leesar MA, Stoddard MF, Dawn B, Jasti VG, Masden R, Bolli R

(2001) Delayed preconditioning-mimetic action of nitroglycerin

in patients undergoing coronary angioplasty. Circulation 103(24):

2935–2941

5. Crisafulli A, Melis F, Tocco F, Pittau G, Lorrai L, Gori T,

Mancardi D, Concu A, Pagliaro P (2007) Delayed precondition-

ing-mimetic actions of exercise or nitroglycerin do not affect

haemodynamics and exercise performance in trained or sedentary

individuals. J Sports Sci 25(12):1393–1401. doi:10.1080/026404

10601128914

6. Gori T, Di Stolfo G, Sicuro S, Dragoni S, Lisi M, Forconi S,

Parker JD (2007) Nitroglycerin protects the endothelium

from ischaemia and reperfusion: human mechanistic insight. Br J

Clin Pharmacol 64(2):145–150. doi:10.1111/j.1365-2125.2007.

02864.x

7. Dragoni S, Gori T, Lisi M, Di Stolfo G, Pautz A, Kleinert H,

Parker JD (2007) Pentaerythrityl tetranitrate and nitroglycerin,

but not isosorbide mononitrate, prevent endothelial dysfunction

induced by ischemia and reperfusion. Arterioscler Thromb Vasc

Biol 27(9):1955–1959. doi:10.1161/ATVBAHA.107.149278

8. Gori T, Dragoni S, Di Stolfo G, Sicuro S, Liuni A, Luca MC,

Thomas G, Oelze M, Daiber A, Parker JD (2010) Tolerance to

nitroglycerin-induced preconditioning of the endothelium: a

human in vivo study. Am J Physiol Heart Circ Physiol 298(2):

H340–H345. doi:10.1152/ajpheart.01324.2008

9. Csont T (2010) Nitroglycerin-induced preconditioning: interac-

tion with nitrate tolerance. Am J Physiol Heart Circ Physiol

298(2):H308–H309. doi:10.1152/ajpheart.01079.2009

10. Schnorbus B, Schiewe R, Ostad MA, Medler C, Wachtlin D,

Wenzel P, Daiber A, Munzel T, Warnholtz A (2010) Effects of

pentaerythritol tetranitrate on endothelial function in coronary

artery disease: results of the PENTA study. Clin Res Cardiol

99(2):115–124. doi:10.1007/s00392-009-0096-z

11. Jurt U, Gori T, Ravandi A, Babaei S, Zeman P, Parker JD (2001)

Differential effects of pentaerythritol tetranitrate and nitroglyc-

erin on the development of tolerance and evidence of lipid

peroxidation: a human in vivo study. J Am Coll Cardiol 38(3):

854–859

12. Daiber A (2010) Redox signaling (cross-talk) from and to mito-

chondria involves mitochondrial pores and reactive oxygen

species. Biochim Biophys Acta. doi:10.1016/j.bbabio.2010.01.032

13. Chen CH, Budas GR, Churchill EN, Disatnik MH, Hurley TD,

Mochly-Rosen D (2008) Activation of aldehyde dehydrogenase-2

reduces ischemic damage to the heart. Science 321(5895):

1493–1495. doi:10.1126/science.1158554

14. Bartoli G, Menegaz G, Lisi M, Di Stolfo G, Dragoni S, Gori T

(2008) Model-based analysis of flow-mediated dilation and

intima-media thickness. Int J Biomed Imaging 2008:738545. doi:

10.1155/2008/738545

15. Edgell CJ, McDonald CC, Graham JB (1983) Permanent cell line

expressing human factor VIII-related antigen established by

hybridization. Proc Natl Acad Sci USA 80(12):3734–3737

16. Wenzel P, Hink U, Oelze M, Seeling A, Isse T, Bruns K,

Steinhoff L, Brandt M, Kleschyov AL, Schulz E, Lange K,

Weiner H, Lehmann J, Lackner KJ, Kawamoto T, Munzel T,

Daiber A (2007) Number of nitrate groups determines reactivity

and potency of organic nitrates: a proof of concept study in

ALDH-2-/- mice. Brit J Pharmacol 150:526–533

17. Beretta M, Sottler A, Schmidt K, Mayer B, Gorren AC (2008)

Partially irreversible inactivation of mitochondrial aldehyde

dehydrogenase by nitroglycerin. J Biol Chem 283(45):30735–

30744. doi:10.1074/jbc.M804001200

18. Ostad MA, Nick E, Paixao-Gatinho V, Schnorbus B, Schiewe R,

Tschentscher P, Munzel T, Warnholtz A (2010) Lack of evidence

for pleiotropic effects of clopidogrel on endothelial function and

inflammation in patients with stable coronary artery disease:

results of the double-blind, randomized CASSANDRA study.

Clin Res Cardiol 100(1):29–36. doi:10.1007/s00392-010-

0199-6

19. Depre C, Park JY, Shen YT, Zhao X, Qiu H, Yan L, Tian B,

Vatner SF, Vatner DE (2010) Molecular mechanisms mediating

preconditioning following chronic ischemia differ from those in

classical second window. Am J Physiol Heart Circ Physiol

299(3):H752–H762. doi:10.1152/ajpheart.00147.2010

20. Shen YT, Depre C, Yan L, Park JY, Tian B, Jain K, Chen L,

Zhang Y, Kudej RK, Zhao X, Sadoshima J, Vatner DE, Vatner SF

(2008) Repetitive ischemia by coronary stenosis induces a novel

window of ischemic preconditioning. Circulation 118(19):1961–

1969. doi:10.1161/CIRCULATIONAHA.108.788240

21. Griesberger M, Kollau A, Wolkart G, Wenzl MV, Beretta M,

Russwurm M, Koesling D, Schmidt K, Gorren AC, Mayer B

(2011) Bioactivation of pentaerythrityl tetranitrate by mitochon-

drial aldehyde dehydrogenase. Mol Pharmacol 79(3):541–548.

doi:10.1124/mol.110.069138

22. Gori T, Munzel T (2009) Symptomatic and hemodynamic benefit

of pentaerythrityl tetranitrate and hydralazine in a case of con-

gestive heart failure. Clin Res Cardiol 98(10):677–679. doi:

10.1007/s00392-009-0053-x

23. Daiber A, Wenzel P, Oelze M, Munzel T (2008) New insights

into bioactivation of organic nitrates, nitrate tolerance and cross-

tolerance. Clin Res Cardiol 97(1):12–20. doi:10.1007/s00392-

007-0588-7

24. Gori T, Daiber A (2009) Non-hemodynamic effects of organic

nitrates and the distinctive characteristics of pentaerithrityl

tetranitrate. Am J Cardiovasc Drugs 9(1):7–15. doi:10.2165/

00129784-200909010-00002

25. Hink U, Daiber A, Kayhan N, Trischler J, Kraatz C, Oelze M,

Mollnau H, Wenzel P, Vahl CF, Ho KK, Weiner H, Munzel T

(2007) Oxidative inhibition of the mitochondrial aldehyde

dehydrogenase promotes nitroglycerin tolerance in human blood

vessels. J Am Coll Cardiol 50(23):2226–2232. doi:10.1016/

j.jacc.2007.08.031

26. Szocs K, Lassegue B, Wenzel P, Wendt M, Daiber A, Oelze M,

Meinertz T, Munzel T, Baldus S (2007) Increased superoxide

production in nitrate tolerance is associated with NAD(P)H oxi-

dase and aldehyde dehydrogenase 2 downregulation. J Mol Cell

Cardiol 42(6):1111–1118. doi:10.1016/j.yjmcc.2007.03.904

27. Chen Z, Foster MW, Zhang J, Mao L, Rockman HA, Kawamoto

T, Kitagawa K, Nakayama KI, Hess DT, Stamler JS (2005) An

essential role for mitochondrial aldehyde dehydrogenase in

nitroglycerin bioactivation. Proc Natl Acad Sci USA 102(34):

12159–12164

28. Ciccone MM, Iacoviello M, Puzzovivo A, Scicchitano P, Moni-

tillo F, De Crescenzo F, Caragnano V, Sassara M, Quistelli G,

Guida P, Favale S (2011) Clinical correlates of endothelial

function in chronic heart failure. Clin Res Cardiol 100(6):

515–521. doi:10.1007/s00392-010-0275-y

29. Trojnarska O, Szczepaniak-Chichel L, Mizia-Stec K, Gabriel M,

Bartczak A, Grajek S, Gasior Z, Kramer L, Tykarski A (2011)

Vascular remodeling in adults after coarctation repair: impact of

descending aorta stenosis and age at surgery. Clin Res Cardiol

100(5):447–455. doi:10.1007/s00392-010-0263-2

30. Zankl AR, Ivandic B, Andrassy M, Volz HC, Krumsdorf U,

Blessing E, Katus HA, Tiefenbacher CP (2010) Telmisartan

improves absolute walking distance and endothelial function in

patients with peripheral artery disease. Clin Res Cardiol

99(12):787–794. doi:10.1007/s00392-010-0184-0

31. Bulut D, Scheeler M, Niedballa LM, Miebach T, Mugge A (2011)

Effects of immunoadsorption on endothelial function, circulating

endothelial progenitor cells and circulating microparticles in

458 Clin Res Cardiol (2012) 101:453–459

123

patients with inflammatory dilated cardiomyopathy. Clin Res

Cardiol 100(7):603–610. doi:10.1007/s00392-011-0287-2

32. Karthikeyan VJ, Blann AD, Baghdadi S, Lane DA, Gareth Bee-

vers D, Lip GY (2011) Endothelial dysfunction in hypertension in

pregnancy: associations between circulating endothelial cells,

circulating progenitor cells and plasma von Willebrand factor.

Clin Res Cardiol 100(6):531–537. doi:10.1007/s00392-010-

0277-9

33. Czucz J, Cervenak L, Forhecz Z, Gombos T, Pozsonyi Z, Kunde

J, Karadi I, Janoskuti L, Prohaszka Z (2011) Serum soluble

E-selectin and NT-proBNP levels additively predict mortality in

diabetic patients with chronic heart failure. Clin Res Cardiol

100(7):587–594. doi:10.1007/s00392-011-0283-6

34. Wenzel P, Schulz E, Gori T, Ostad MA, Mathner F, Schildknecht

S, Gobel S, Oelze M, Stalleicken D, Warnholtz A, Munzel T,

Daiber A (2009) Monitoring white blood cell mitochondrial

aldehyde dehydrogenase activity: implications for nitrate therapy

in humans. J Pharmacol Exp Ther 330(1):63–71. doi:10.1124/

jpet.108.149716

35. Weber W, Michaelis K, Luckow V, Kuntze U, Stalleicken D

(1995) Pharmacokinetics and bioavailability of pentaerithrityl

tetranitrate and two of its metabolites. Arzneimittelforschung

45(7):781–784

36. Kanamasa K, Naito N, Morii H, Nakano K, Tanaka Y, Kitayama

K, Haku R, Kai T, Yonekawa O, Nagatani Y, Ishikawa K (2004)

Eccentric dosing of nitrates does not increase cardiac events in

patients with healed myocardial infarction. Hypertens Res

27(8):563–572 (pii:JST.JSTAGE/hypres/27.563)

37. Poss J, Jacobshagen C, Ukena C, Bohm M (2011) Hotlines and

clinical trial updates presented at the German Cardiac Society

Meeting 2010: FAIR-HF, CIPAMI, LIPSIA-NSTEMI, Handheld-

BNP, PEPCAD III, remote ischaemic conditioning, CERTIFY,

PreSCD-II, German Myocardial Infarction Registry, DiaRegis.

Clin Res Cardiol 99(7):411–417. doi:10.1007/s00392-010-0176-0

Clin Res Cardiol (2012) 101:453–459 459

123