Pharmacology & Pharmacy, 2011, 2, 347-353
doi:10.4236/pp.2011.24045 Published Online October 2011 (http://www.SciRP.org/journal/pp)
347
Vazoactive Effects of Oxidative Stress Elicited by
Hydrogen Peroxide in the Human Umbilical
Artery: An in Vitro Study
Ipek Duman*, Necdet Dogan
Department of Pharmacology, Meram Faculty of Medicine, Selcuk University, Konya, Turkey.
Received June 5th, 2011; revised July 20th, 2011; accepted August 30th, 2011.
ABSTRACT
The vasoactive effects of oxidative stress induced by hydrogen peroxide (H2O2) on human umbilical artery strips as well
as the possible mechanisms involved are studied. Contraction responses to cumulative H2O2 (10–7 M - 3 × 10–2 M) in
endothelium intact and denuded umbilical arteries and responses to cumulative H2O2 after incubation with L-NAME
(10–4 M) (n = 8), indomethacin (10–5 M) (n = 8) and verapamil (10–6) (n = 8) were recorded. Responses elicited with
cumulative H2O2 in Ca2+ free extracellular medium and the responses to cumulative Ca2+ (10–4 M - 2 × 10–3 M) after
H2O2 (10–3 M) induced contraction were also studied. The Emax for each experiment was calculated. p < 0.05 was con-
sidered as significant. H2O2 elicited contraction was greater in endothelium denuded artery strips compared to endo-
thelium intact strips (p < 0.05). Compared to control, incubation with L-NAME significantly augmented (p < 0.05),
while verapamil and indomethacin inhibited the contractions elicited by cumulative H2O2 (p < 0.05). Ca2+ free ex-
tracellular medium caused decreases in cumulative H2O2 elicited contractions and cumulative Ca2+ caused concentra-
tion dependent increases in the contraction caused by a single bolus of H2O2 (p < 0.05). Exposure to H2O2 causes con-
centration-dependent constriction in human umbilical arteries. The presence of the endothelium and NOS enzyme acti-
vation influences the H2O2 responses. Removal of the endothelium increases the H2O2 elicited contractions more than
incubation with L-NAME suggesting beside NO, other endothelial vasodilators are also involved in vascular tonus of
the umbilical arteries. Both intracellular and extracellular Ca2+ ions and constrictor cyclooxygenase metabolites play a
role in the contractile responses elicited by H2O2 in human umbilical arteries.
Keywords: Umbilical Arteries, Hydrogen Peroxide, Indomethacin, L-NAME, Oxidative Stress, Pre-Eclampsia, Reactive
Oxygen Species, Verapamil
1. Introduction
Pre-eclampsia (PE) is a major cause of fetal growth re-
striction and perinatal complications. In PE, there is in-
creased resistance to placental circulation which leads to
reduced uteroplacental blood flow followed by placental
dysfunction and intrauterine fetal growth restriction [1,2].
Umbilical blood vessels are not innervated therefore the
control of umbilical blood flow depends entirely on
vasoreactive substances either released locally or pre-
sented in the circulation [3]. Pregnancy is a state of oxi-
dative stress, characterized by the placental production of
reactive oxygen species (ROS) including superoxide and
hydrogen peroxide (H2O2) [4,5]. It is considered that
during normal pregnancy, the rate of production of ROS
is offset by their elimination by abundant antioxidant
defenses. However in PE and preterm labor, due to ex-
cessive oxidative stress and lipid peroxidation (LPO)
ROS overpowers antioxidant defenses, leading to reduc-
tion of uteroplacental blood flow [5,6]. H2O2 is a power-
ful by product of LPO and is used as a model of oxida-
tive stress. H2O2 easily crosses cell membranes and lead
to cellular oxidative damage. LPO has been studied in-
tensively over decades and remains to be a hot topic in
biological research but still there is little information on
the effects of LPO on human umbilical arteries [7-9].
This in vitro study was designed to assess the effects of
H2O2 used as a model of oxidative stress on human um-
bilical arteries as well as the possible mechanisms in-
volved.

Vazoactive Effects of Oxidative Stress Elicited by Hydrogen Peroxide in the 348
Human Umbilical Artery: An in Vitro Study
2. Materials and Methods
The University Human Ethics Committee approved this
study. All umbilical cords used in the experiments were
remnant tissues, which would have otherwise been dis-
carded.
2.1. Sample Collection
After maternal consent, human umbilical cords were col-
lected from healthy full-term normal deliveries. After
delivery, the umbilical cord was clamped at both placen-
tal and fetal ends. An untouched 15 - 20 cm long seg-
ment of the umbilical cord was taken from the placental
side within 10 min of delivery and placed in cold Krebs-
Henseleit solution for transport to the laboratory.
2.2. Blood Vessel Preparation
Umbilical arteries were separated from the surroundings
in warm modified Krebs-Henseleit solution. The isolated
artery was cut spirally to form 2 - 3 mm wide and 15 - 20
mm long strips. The strips were suspended between two
stainless steel hooks in organ baths (10 ml) containing
Krebs-Henseleit buffer maintained at 37˚C. One hook
was anchored onto the organ bath and the other was
connected to a movable transducer (Model FT 03, Grass
Instrument Co. MA, USA) and a polygraph (Model 7,
Grass Instrument Co. MA, USA) for measurement and
recording of changes in isometric tension.
2.3. Experimental Protocols
Protocols were conducted with endothelium intact artery
strips except for protocol 1 which used both endothelium
intact and endothelium denuded artery strips. Endothe-
lium removal was done by gently denuding the endothe-
lium of the artery with cotton swabs. The integrity of the
endothelium was tested by, first pre-contracting the de-
nuded strips with serotonin (10–4 M), and then adding
acetylcholine (10–6 M) before each experiment. Removal
of the endothelium was confirmed if the vessels con-
tracted in response to acetylcholine.
Strips were aerated with a gas mixture of 95% O2:5%
CO2 throughout the experiment. Strips were initially
placed under a resting tension of 1 g and were allowed to
equilibrate for one hour. During this period the bath solu-
tion was changed every 15 minutes and the resting ten-
sion was readjusted to the 1 g level. Following the
washout period, the initial control contraction of the
strips to serotonin (10–4 M) was recorded. The strips were
washed again with the buffer solution and allowed to rest.
After an equilibrium period, the following procedures
were conducted at 37˚C:
Protocol 1: to determine the role of the endothelium
on the reactivity of the human umbilical artery to H2O2,
umbilical artery strips both with intact endothelium (n =
8) and denuded endothelium (n = 8) were used. The
strips precontracted with serotonin (10–4 M) were sub-
jected to cumulative doses of H2O2 (10–7 M - 3 × 10–2 M)
at resting tension and concentration-response curves
were obtained. Further protocols were conducted with
endothelium intact artery strips.
Protocol 2: to determine the role of nitric oxide (NO)
in the mechanism of H2O2 elicited contractions; umbili-
cal artery strips precontracted with serotonin (10–4 M)
were incubated for 20 minutes with a NO synthase inhibi-
tor, Nω-nitro-L-arginine methyl ester (L-NAME) (10–4 M)
(n = 8) After this incubation, concentration-response cur-
ves were obtained to cumulative H2O2 (10–7 M - 3 × 10–2
M).
Protocol 3: to determine the role of prostanoids in the
mechanism of H2O2 elicited contractions; umbilical ar-
tery strips precontracted with serotonin (10–4 M) were
incubated for 20 minutes with a cyclooxygenase inhibitor,
indomethacin (10–5 M) (n = 8). After this incubation,
concentration-response curves were obtained to cumula-
tive H2O2 (10–7 M - 3 × 10–2 M).
Protocol 4: to determine the role of Ca2+ channels in
the mechanism of H2O2 elicited contractions; umbilicalk
artery strips precontracted with serotonin (10–4 M) were
incubated for 20 minutes with a Ca2+ channel blocker,
verapamil (10–6 M) (n = 8). After this incubation, con-
centration-response curves were obtained to cumulative
H2O2 (10–7 M - 3 × 10–2 M).
Protocol 5: the effects of Ca2+ on H2O2 elicited re-
sponses were studied in another group of umbilical arter-
ies (n = 8). The artery strips were allowed to rest in modi-
fied Ca2+ free Krebs-Henseleit solution containing 1 mM
of ethyleneglycol-bis-(β-aminoethyl ether) N’tetraacetic
acid (EGTA) for 60 min, which was changed every 15
min. Cumulative H2O2 (10–7 M - 3 × 10–2 M) was added
to the organ bath and concentration-response curves were
obtained. In a different group of umbilical strips (n = 8),
the strips were allowed to rest in modified Ca2+ free
Krebs-Henseleit solution containing 1mM of EGTA for
60 min, which was changed every 15 min. After obtain-
ing a contraction curve with a bolus of H2O2 (10–3 M),
cumulative Ca2+ (10–4 M - 2 × 10–3 M) was added to the
organ bath and concentration-response curves were ob-
tained.
2.4. Materials
H2O2, magnesium sulphate (MgSO4), potassium hydro-
gen phosphate (KH2PO4) sodium bicarbonate (NaHCO3),
potassium chloride (KCl), sodium chloride (NaCl), and
calcium chloride (CaCl2) were obtained from Merck

Vazoactive Effects of Oxidative Stress Elicited by Hydrogen Peroxide in the 349
Human Umbilical Artery: An in Vitro Study
(Merck KGaA, Darmstadt, Germany) and serotonin hy-
drochloride, L-NAME, indomethacin, verapamil and
EGTA were purchased from Sigma Chemical Co. (St.
Louis, MO, U.S.A.). Krebs-Henseleit solution and modi-
fied Krebs-Henseleit without calcium were prepared in
the laboratory with compositions of (in mM) NaCl 119;
KCl 4.7; MgSO4 1.5; KH2PO4 1.2; CaCl2 2.5; NaHCO3
25; glucose 11, and NaCl 119; KCl 4.7; MgSO4 1.5;
KH2PO4 1.2; NaHCO3 25; glucose 11, EGTA 1 respec-
tively. All agents were dissolved in distilled water.
2.5. Data and Statistical Analysis
The contraction is expressed as percentage (%) of the
contractile level that was induced by serotonin. The Emax
(% of maximum contraction) in each group and pD2 (the
negative logarithm of the concentration which elicits
50% contraction) for groups in which Emax > 50% were
calculated. All results are expressed as the mean ± stan-
dard deviation of mean and n denotes the number of hu-
man umbilical cords which the arterial strips were ob-
tained. Analysis of variance (ANOVA) and Tukey’s
HSD tests were used were appropriate to determine the
differences between the percentage values using a com-
puter statistical package (SPSS, Chicago, IL, USA). p <
0.05 was considered as significant.
3. Results
3.1. The Effect of Endothelium on H2O2 Elicited
Contractions
H2O2 (10–7 M - 3 × 10–2 M) elicited concentration de-
pendent contraction in isolated human umbilical artery
strips both with (Emax = 63.5 ± 3.7, pD2 = 3.08 ± 0.1) and
without (Emax = 101.8 ± 9.6, pD2 = 4.10 ± 0.26) endothe-
lium. There were significant differences among these
strips in terms of Emax and pD2 values with significantly
larger contractions in the endothelium denuded strips (p
< 0.05) (Figure 1).
3.2. The Effect of L-NAME, Indomethacin and
Verapamil Incubation on H2O2 Elicited
Contractions
Compared to control (Emax = 63.5 ± 3.7, pD2 = 3.08 ±
0.0), incubation with L-NAME significantly augmented
(Emax = 91.8 ± 8.3, pD2 = 3.74 ± 0.2) (p < 0.05), while
verapamil (Emax = 25.1 ± 3.3) and indomethacin (Emax =
14.1 ± 3.4) significantly inhibited the contractions elic-
ited by cumulative H2O2 (p < 0.05) (Figure 2).
3.3. The Effect of Ca2+ on H2O2 Elicited
Contractions
When compared with the maximum contraction responses
Figure 1. Concentration-response curves for cumulative H2O2
(10–7 M - 3 × 10–2 M) on isolated human umbilical artery
strips with and without endothelium. Data expressed as the
percentage of the control contractile response elicited by 10–4
M of serotonin. Mean ± SD (n = 8).
Figure 2. Concentration-response curves for cumulative H2O2
(10–7 M - 3 × 10–2 M) on isolated human umbilical artery
strips with endotheli um after incubation with L-NAME (10 –4
M), verapamil (10–6 M) and indomethacin (10–5 M). Data
expressed as the percentage of the control contractile re-
sponse elicited by 10–4 M of serotonin. Mean ± SD (n = 8).
with Krebs-Henseleit solution (Emax = 63.5 ± 3.7, pD2 =
3.08 ± 0.0), providing a Ca2+ free extracellular medium
caused significant decreases in cumulative H2O2 elicited
contractions (Emax = 38.7 ± 5.8) (p < 0.05) (Figure 3).
After resting in modified Ca2+ free Krebs-Henseleit solu-
tion, cumulative Ca2+ (10–4 M - 2 × 10–3 M) caused sig-
nificant concentration dependent increases (Emax = 98.5 ±
6.1) in the contraction caused by a single bolus of H2O2
(Emax = 23.3 ± 3.3) (p < 0.05) (Figure 4).

Vazoactive Effects of Oxidative Stress Elicited by Hydrogen Peroxide in the 350
Human Umbilical Artery: An in Vitro Study
Figure 3. Concentration-response curves for cumulative
Ca2+ (10–4 M - 2 × 10–3 M) on H2O2 (10–3 M) induced con-
striction of human umbilical arteries. Data expressed as the
percentage of the control contractile response elicited by
10–4 M of serotonin. Mean ± SD (n = 8).
Figure 4. Concentration-response curves for cumulative
H2O2 (10–7 M - 3 × 10–2 M) on isolated human umbilical ar-
tery strips in Krebs-Henseleit solution and modified Krebs-
Henseleit without calcium. Data expressed as the percent-
age of the control contractile response elicited by 10–4 M of
serotonin. Mean ± SD (n = 8).
4. Discussion
The present study demonstrates that in vitro experimental
model of oxidative stress elicited by H2O2 causes con-
centration dependent vasoconstriction in human umbili-
cal arteries both with and without endothelium. Both
intracellular and extracellular Ca2+ ions and cyclooxy-
genase enzyme activation play a role in the contractile
responses elicited by H2O2 in human umbilical arteries.
The presence of the endothelium and NOS enzyme acti-
vation influences the H2O2 responses.
The known vasoactive substances responsible for the
control of umbilical flow include local vasoconstrictors
such as endothelin-1, thromboxane A2 and prostaglandin
F2α and also vasodilators such as prostacyclin (PGI2),
nitric oxide (NO) and end8othelium derived hyperpolar-
izing factor (EDHF) [10,11]. It is known that under oxi-
dative stress conditions such as pre-eclampsia placental
vascular resistance increases [12,13]. The mechanisms
by which ROS cause vasoconstriction are incompletely
understood. At a vascular level, endothelial cells are a
target for, and a source of, H2O2. Previous work with
different species and vascular structures yielded con-
flicting results. Similar to our study, H2O2 caused vaso-
constriction in human umbilical artery [14,15], pulmo-
nary artery [16] and rat aorta [17]. Contrary to these re-
sults, H2O2 caused vasodilation in rabbit [18], rat mese n -
teric artery [19], guinea-pig aorta [20] and porcine coro-
nary artery [21]. The above results lead us to think that
the vasoactive properties of H2O2 vary with species, tis-
sue and experimental conditions [20,22]. Therefore iso-
lated human placental arteries and veins should be con-
sidered most suitable for in-vitro PE models.
In the present study our data suggest that cumulative
H2O2 causes more potent vasoconstriction in endothelium
denuded human umbilical arteries. This effect may be
attributed to the loss of vasodilators produced by the en-
dothelium such as NO, EDHF and PGI2. Similar results
have been shown previously in the rat aorta [17,22].
Rodriguez-Martinez et al. [17] have demonstrated that
oxygen-derived free radicals are involved in the contrac-
tile effects of H2O2; endothelium protects against oxida-
tive injury caused by H2O2 in smooth muscle cells, en-
dothelial NO has a protective role on the contractile ef-
fect induced by H2O2 in normotensive rats; and this pro-
tective role of endothelial NO is lost under oxidative
stress such as hypertension. The role of ROS and NO in
the pathogenesis of pre-eclampsia using human umbilical
vein endothelial cell cultures, have been investigated by
Matsubara et al. [9] in normal and pre-eclampsia patients.
They argued that endothelial dysfunction during PE pos-
sibly results from the inactivation of NO by superoxide
ions. This is supported by our results, in which umbilical
artery strips incubated with L-NAME, which is a NO
synthase inhibitor, significantly increased the contraction
elicited by H2O2. Furthermore, complete endothelial re-
moval resulted in higher vasoconstriction than the L-
NAME incubated human umbilical arteries. This implies
that besides NO, other endothelial factors such as EDHF
and PGI2 may be involved in the process. Together with
previous literature, these results lead us to think that NO

Vazoactive Effects of Oxidative Stress Elicited by Hydrogen Peroxide in the 351
Human Umbilical Artery: An in Vitro Study
acts as a negative modulator against H2O2 elicited con-
tractions in placental arteries and also may be protective
against oxidative insult [17,19,22].
Lelung et al. [3] have demonstrated that sodium nitro-
prusside, an exogenous source of NO, only elicits small
significant relaxations in isolated human umbilical arter-
ies. They are in the opinion that NO might not be the
major mediator responsible for vasodilation in umbilical
arteries. Previous work also suggests that, instead of NO
prostacyclin was the main factor mediating the endothe-
lium dependent relaxation in human umbilical arteries
[10,14,23]. Klockenbush et al. [24] have suggested that
rather than NO, prostacyclin plays a major role in the
vasoreactivity of umbilical and fetal circulation. H2O2
elicited vasoreactivity can be relaxation or constriction
depending on the basal tonus of isolated pulmonary ar-
teries, and these effects are mediated by phospholipase
A2 activation leading to prostacyclin or thromboxane A2
release [25]. In the present study the H2O2 elicited con-
traction in umbilical arteries were significantly inhibited
in the presence of indomethacin, a cyclooxygenase en-
zyme inhibitor. These data suggest that increased con-
strictor cyclooxygenase metabolites as a result of in-
creased arachidonic acid metabolism partially mediate
the constrictive effects of H2O2 in the vascular smooth
muscle of human umbilical arteries. Previous research
shows that prostaglandin H2 and more possibly throm-
boxane A2 are involved in the contractile response [26,
27]. These findings are in accordance with studies which,
showed that H2O2 increases prostaglandin F2α and
thromboxane A2 in rat aorta smooth muscle [28,29].
Ca2+ is essential for the contraction of smooth muscles
and it is believed that intracellular Ca2+ homeostasis
plays a major role in antioxidant activity [30-33]. The
harmful effects of ROS can influence the ion channels or
ion pumps, which maintain low Ca2+ levels under normal
conditions [33,34]. It is known from previous work that
in different tissues H2O2 can increase intracellular Ca2+
by promoting mobilization of Ca2+ and Ca2+ influx [31,33,
34]. In isolated rat cardiomyocytes Gen et al. [33] have
shown that H2O2 increases intracellular Ca2+ in a dose
dependent manner. In our study we have found that H2O2
induced contractions increase in a dose dependent pattern
when cumulative Ca2+ was added after a single bolus of
H2O2 in human umbilical arteries.
It has been reported that NO modulates Ca2+-channel
activity in vascular smooth muscle and induces relaxa-
tion [35]. Incubation with verapamil, a Ca2+-channel an-
tagonist significantly decreased but did not totally block
H2O2 induced contractions. Sotnikova [22] and Yang et
al. [36] have reported similar results in rat aorta with
calcium antagonists. In the present study, both incubation
with verapamil and resting the arteries in Ca2+-free me-
dium attenuated but did not prevent H2O2 induced con-
tractions. These findings suggest that both intracellular
and extracellular Ca2+ mediate these contractions. It has
been suggested previously that the decrease caused by
ROS on membrane resistance may depolarize the cells
and thus activate voltage sensitive Ca2+ channels and lead
to an increase in intracellular Ca2+ [31,32]. Also, Ca2+
channel blockers are thought to be ineffective in blocking
mobilization of Ca2+ from intracellular stores but effec-
tively block the influx of extracellular Ca2+ via the L type
Ca2+ channels [33].
5. Conclusions
In conclusion, in this in-vitro model for oxidative stress
we have demonstrated that exposure to H2O2 causes
concentration-dependent constriction in human umbilical
arteries. Removal of the endothelium increased the H2O2
elicited contractions more than incubation with L-NAME
suggesting beside NO, other endothelial vasodilators are
also involved in vascular tonus of the umbilical arteries.
Inhibition of H2O2 elicited contractions with indometha-
cin suggests that increased constrictor cyclooxygenase
metabolites partially mediate the constrictive effects of
H2O2 in the vascular smooth muscle of human umbilical
arteries. Incubation with verapamil, and Ca2+ free me-
dium significantly decreased but did not totally block
H2O2 induced contractions suggesting both intracellular
and extracellular Ca2+ involvement in H2O2 elicited con-
tractions.
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