Vazoactive Effects of Oxidative Stress Elicited by Hydrogen Peroxide in the Human Umbilical Artery: An <i>in Vitro</i> Study

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Pharmacology & Pharmacy, 2011, 2, 347-353

doi:10.4236/pp.2011.24045 Published Online October 2011 (http://www.SciRP.org/journal/pp)

Copyright © 2011 SciRes. 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.

Email: *[email protected]

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

Copyright © 2011 SciRes. PP

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).

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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

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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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