Maternal smoking and impaired endothelium-dependent nitric oxide–mediated relaxation of uterine small arteries in vitro

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Maternal smoking and impaired endothelium-dependent nitric oxide–mediated relaxation of uterine small arteries in vitro Malene R. Andersen, MSc, PhD; Niels Uldbjerg, MD, DMSc; Steen Stender, MD, DMSc; Puk Sandager, MD, PhD; Christian Aalkjær, MD, DMSc OBJECTIVE: This study aimed to investigate the endothelium-depen-

dent relaxation of uterine small arteries from pregnant nonsmokers, smokers, and ex-smokers who stopped smoking early in pregnancy. STUDY DESIGN: Uterine arteries were dissected from myometrial biop-

sies obtained during elective cesarean sections of 34 uncomplicated, singleton pregnancies, and the vascular function was assessed in a wire myograph for isometric recordings. Serum cotinine verified selfreported smoking; 15 were nonsmokers, 10 were smokers, and 9 were ex-smokers. RESULTS: Arteries from smokers and ex-smokers had reduced brady-

kinin-induced relaxation compared to arteries from nonsmokers (P ⬍

.05). The relaxation response to the nitric oxide donor sodium nitroprusside was similar in arteries from nonsmokers and smokers but was better in arteries from ex-smokers (P ⬍ .05). CONCLUSION: The findings suggest that maternal smoking reduces

endothelium-dependent nitric oxide–mediated relaxation in uterine small arteries and that smoking cessation early in pregnancy does not fully abolish this deleterious effect, despite improvement in relaxation to nitroprusside. Key words: bradykinin, endothelium, nitric oxide, pregnancy, smoking

Cite this article as: Andersen MR, Uldbjerg N, Stender S, et al. Maternal smoking and impaired endothelium-dependent nitric oxide–mediated relaxation of uterine small arteries in vitro. Am J Obstet Gynecol 2011:204;177.e1-7.

W

omen who continue to smoke during pregnancy deliver infants with a smaller birthweight than those of nonsmokers.1-6 This negative effect on birthweight is, however, abolished by smoking cessation early in pregnancy.4-6 Since the effect of smoking on birth-

From the Department of Clinical Biochemistry (Drs Andersen and Stender), Copenhagen University Hospital, Gentofte, Denmark; the Department of Obstetrics and Gynecology (Drs Andersen, Uldbjerg, and Sandager), Aarhus University Hospital, Skejby; and the Institute of Physiology and Biophysics (Dr Aalkjær), Aarhus University, Aarhus, Denmark. Received June 3, 2010; revised Aug. 11, 2010; accepted Sept. 13, 2010. Reprints not available from the authors. This study was supported by grants from the Faculty of Health Sciences (Aarhus University, SUN-2002-653); the Clinical Institute (Aarhus University); the Research Foundation of Aarhus University Hospital, Skejby; the Boennelykke Foundation; and the Master Smith Niels Hansen and Wife Johanne, born Frederiksen Foundation. 0002-9378/$36.00 © 2011 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2010.09.006

weight is still present after correction for gestational age, parity, and infant sex, as well as other sociodemographic and clinical characteristics that may be associated with birthweight, it has been suggested that maternal smoking causes impaired fetal growth.2-4,6 One important factor in maintaining fetal growth is the blood flow in both maternal uterine arteries as well as fetal placental and umbilical vessels, which deliver oxygen and nutrients to the fetus.7-9 In human Doppler ultrasonography studies, the blood flow velocity in different fetal vascular beds was increased in maternal smokers, consistent with a greater vascular resistance and/or a higher blood pressure in the fetus. Surprisingly, however, no effect was found in uterine arteries.10,11 Doppler waveforms reflect but do not necessarily accurately measure the maximal velocity of blood in the direction of the ultrasound beam from which the Doppler signal is produced,10 suggesting that differences in uterine blood flow between nonsmokers and smokers are too small for reliable detection by this method. Previous studies have shown that the uterine blood flow was reduced after intravenous infu-

sion of nicotine doses in pregnant monkeys12 and ewes13,14 resulting in serum nicotine levels comparable to those found in human cigarette smokers, suggesting that this issue should be studied with more sensitive methods. In nonpregnant healthy human beings, cigarette smoking15-17 and intravenous infusion of nicotine18 were found to impair endothelium-dependent relaxation mediated by vasodilator nitric oxide (NO). NO deficiency may be related to intrauterine growth restriction. For instance, a lower NO-dependent flow-mediated dilation in the brachial artery and a higher plasma level of asymmetric dimethylarginine (an inhibitor of endothelial NO synthase [eNOS] that competes with L-arginine),19 as well as lower eNOS expression in the umbilical artery20 and lower eNOS activity in placental villous tissue,21 were found in women whose pregnancies were complicated by intrauterine growth restriction compared to normal outcome pregnancies. In addition, we recently showed that the newborns of smoking mothers were smaller as measured by weight and head circumference and had lower vascular eNOS activity and concentration com-

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pared to the newborns of nonsmoking mothers2; further, smoking cessation early in pregnancy abolished these effects.6 To address the mechanisms through which growth restriction may occur, in the current study we have investigated uterine small artery function in vitro under controlled conditions. Thus, the present study was designed to evaluate the role of the endothelium-derived vasodilators in uterine small arteries from maternal nonsmokers, smokers, and exsmokers. Self-reported smoking was validated by serum cotinine.

M ATERIALS AND M ETHODS Study participants A total of 49 randomly selected Caucasian women with uncomplicated, singleton pregnancies who were admitted to the Department of Obstetrics and Gynecology, Aarhus University Hospital, Skejby, were invited to participate in the study (February 2004 through October 2006). For inclusion in the study, women who were scheduled for term elective cesarean section had to fit 1 of 3 categories: (1) lifelong nonsmokers; (2) smokers who smoked at least 5 cigarettes/d; or (3) ex-smokers who stopped smoking during the first trimester. Women with cardiovascular disease, diabetes mellitus, gestational diabetes, and preeclampsia, as well as those who delivered at ⬍37 completed weeks of gestation, were excluded from the study. The women answered a questionnaire at ⬃11 weeks of gestation to provide information about lifestyle and sociodemographic factors. The nature and purpose of the study were explained verbally and in writing to each woman 1-3 days before surgery at ⬃39 weeks of gestation, and written consent and information on smoking habits were obtained from those who agreed to participate. The investigation conformed to the principles outlined in the Declaration of Helsinki and was approved by the Scientific Ethics Committee (1988/1349, 1991/2060, and 20030274) and the Danish Data Surveillance Authority (2009-41-3861). Of the 49 invited women, 42 fulfilled the inclusion criteria and agreed to par177.e2

ticipate. During 1 cesarean section the surgeon forgot to obtain a uterine biopsy; in 3 biopsies no arteries were found; in 3 biopsies the arteries had an outer diameter of ⬎500 ␮m; and in 1 biopsy the arteries could not maintain tension throughout the experiments, leaving 34 pregnant women in the main study.

Serum cotinine measurements Cotinine was quantified in maternal serum obtained at ⬃39 weeks of gestation by chemiluminescent immunoassays (DPC Scandinavia, Mölndal, Sweden) with an IMMULITE 2500 analyzer. The detection limit of cotinine in this assay is 10.0 ng/mL. Drugs and solutions All drugs except U46619 and indomethacin were obtained from Sigma Chemical Company, St Louis, MO. U46619 was obtained from Upjohn Company, Kalamazoo, MI, and indomethacin (Confortid) from Dumex Ltd, Copenhagen, Denmark. To prepare stock solutions, the majority of the drugs were dissolved in distilled water. Bradykinin was dissolved in 0.05% bovine serum albumin, and U46619 in absolute ethanol followed by dilution in distilled water. The physiological salt solution (PSS) was composed of the following: 119 mmol/L sodium chloride (NaCl), 4.6 mmol/L potassium chloride (KCl), 15 mmol/L sodium bicarbonate, 1.5 mmol/L calcium chloride, 0.5 mmol/L magnesium chloride, 1.2 mmol/L monosodium phosphate, and 11 mmol/L glucose. The potassium-depolarizing PSS (K-PSS) had the same composition as PSS except that the final concentration of KCl was 124 mmol/L and NaCl was omitted. Artery preparation At elective cesarean section between 8:25 AM and 2:30 PM, a myometrial biopsy specimen (⬃10 ⫻ 5 ⫻ 5 mm) was obtained from the lateral part of the upper edge of the isthmic incision. The biopsy was immediately placed in ice-cold PSS, gassed with 5% carbon dioxide in air (pH 7.4), and transported from the operating room to the laboratory within 15 minutes. From each biopsy, 3 arteries with an outer diameter between 250-500

American Journal of Obstetrics & Gynecology FEBRUARY 2011

www.AJOG.org ␮m were isolated in cold (⬃4°C) PSS by microdissection.

Mounting and normalization of arteries The 3 arteries obtained from each woman were individually mounted on 2 stainless steel wires (40 ␮m in diameter) in 5-mL organ baths of 2 separate 2-channel wire myographs in PSS at room temperature. The arteries were subsequently equilibrated in PSS for 30 minutes at 37°C. To assess artery normalization the relationship between the resting wall tension (force divided by twice the segment length) and internal circumference was determined. These data were used to calculate the internal circumference (IC100) using the Laplace equation,22 which corresponds to a transmural pressure of 100 mm Hg for a relaxed vessel in situ. The arteries were set to IC1 ⫽ 0.9 ⫻ IC100, which provides a maximal active force production of the vessel (data not shown). The normalized lumen diameter (l1) of the arteries was calculated as l1 ⫽ IC1/␲. The arteries of nonsmokers, smokers, and ex-smokers were similar with respect to length, outer diameter measured before mounting in the myograph, and l1 (data not shown). Several studies have demonstrated that cold storage overnight of dissected arteries did not influence the artery function.23-25 In addition, we found no differences in the contraction and relaxation responses if arteries were stored for 1 or 2 days in cold gassed PSS (data not shown).24 Based on these observations the arteries were stored in cold gassed PSS for 1 or 2 days until mounted in a small vessel wire myograph (Myo-interface model 500A; Danish Myo Technology, Aarhus, Denmark). Wire myograph studies The 3 arteries obtained from each woman were successively activated 2-3 times with K-PSS and exposed to study protocol A, B, and C, respectively, as follows: Protocol A The artery was incubated for 20 minutes in PSS. After incubation, the artery was

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lished, a time control for the additions of SNP was conducted (that is the artery was preconstricted with U46619 but SNP was not added).

FIGURE 1

Traces of isometric force of uterine small arteries

Representative traces of wire myograph recordings of isometric force of uterine small arteries from nonsmokers (top), smokers (middle), and time control (bottom). Arteries were preconstricted with U46619 for 10 minutes, followed by cumulative addition of bradykinin (BK) at 3-minute intervals. Tracings show point at which each addition was made, and corresponding cumulative concentration of BK in organ bath is expressed as molar concentration.

Statistical analysis Data are presented as mean ⫾ SEM or number (percent). The tension is expressed as N/m, and relaxation is reported as the percent of the initial contraction. The area under the response curve (AUC) was calculated with the computer program GraphPad Prism 4.03 (GraphPad Software, San Diego, CA). Body mass index before pregnancy and maximum relaxation (Rmax) by bradykinin and SNP were log-transformed for analysis because these data were highly skewed. Continuous data for the nonsmokers, smokers, and ex-smokers were analyzed with the Kruskal-Wallis 1-way analysis of variance by ranks test, followed by 3 pairwise comparisons of the mean ranks with a Bonferroni correction to account for multiple comparisons. Associations of categorical data with smoking status were assessed by the ␹2 test for independence. Statistical calculations were carried out with the Stata 8.2 computer program (StataCorp, College Station, TX), and differences were considered significant at P ⬍ .05.

Andersen. Maternal smoking and uterine vasorelaxation. Am J Obstet Gynecol 2011.

R ESULTS preconstricted with the thromboxane A2 analog U46619 (0.1 ␮mol/L) for 10 minutes, followed by cumulative additions of bradykinin (10 pmol/L-1 ␮mol/L, with additions at 3-minute intervals) (Figure 1, top and middle). Then the bath was washed with 3-5 changes of PSS until the baseline tension was reestablished. This procedure was repeated after incubation for 20 minutes with the nonselective cyclooxygenase (COX) inhibitor indomethacin (10 ␮mol/L), and then with the competitive eNOS inhibitor L-NGnitroarginine (L-NNA) (100 ␮mol/L), and finally with both inhibitors together. Protocol B (time controls) The artery was treated as in protocol A except that no bradykinin was added. This protocol was performed to ensure

that the relaxations observed in protocol A were not due to spontaneous activity (Figure 1, bottom). Protocol C Initially a cumulative concentration response relationship to U46619 (0.1 nmol/L-3 ␮mol/L, with additions at 3-minute intervals) was obtained followed by wash-out of the bath with 3-5 changes of PSS until the baseline tension was reestablished. Then the artery was preconstricted (0.1 ␮mol/L U46619 for 10 minutes) and subsequently exposed to cumulative additions of the NO donor sodium nitroprusside (SNP) (0.1 nmol/ L-10.0 ␮mol/L, with additions at 3-minute intervals). Finally, after the bath was washed with 3-5 changes of PSS until the baseline tension was reestab-

Clinical, sociodemographic, and lifestyle characteristics Table 1 shows that maternal nonsmokers, smokers, and ex-smokers were similar in age, body mass index before pregnancy, blood pressure, parity, years of schooling, and gestational age. All nonsmokers had never smoked, except for one who had stopped smoking ⬃18 years before the pregnancy. The women who kept smoking during pregnancy consumed a 2-fold higher number of cigarettes/d before pregnancy than those who stopped smoking in early pregnancy. During pregnancy, the continual smokers almost halved the number of cigarettes consumed per day (P ⬍ .05 by Mann-Whitney U test). The exsmokers stopped smoking at ⬃6 weeks of gestation. None of the samples from the nonsmokers and ex-smokers had

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TABLE 1

Clinical, sociodemographic, and lifestyle characteristics of pregnant nonsmokers, smokers, and ex-smokers Characteristic

Nonsmokers (n ⴝ 15)

Smokers (n ⴝ 10)

Ex-smokers (n ⴝ 9)

Maternal characteristics

.....................................................................................................................................................................................................................................

Age, y

33 ⫾ 1

33 ⫾ 1

30 ⫾ 2

BMI, kg/m

25 ⫾ 2

26 ⫾ 2

23 ⫾ 1

Systolic BP, mm Hg

117 ⫾ 4

120 ⫾ 4

118 ⫾ 3

Diastolic BP, mm Hg

74 ⫾ 3

69 ⫾ 3

69 ⫾ 3

0 (0)

10 (100)

0 (0)

..................................................................................................................................................................................................................................... 2 ..................................................................................................................................................................................................................................... ..................................................................................................................................................................................................................................... ..................................................................................................................................................................................................................................... a

Cotinine-positive samples, n (%)

.....................................................................................................................................................................................................................................

Cotinine, ng/mL

⬍10

181 ⫾ 41

⬍10

.....................................................................................................................................................................................................................................

Self-reported smoking

........................................................................................................................................................................................................................... b

Before pregnancy, cigarettes/d

0

22 ⫾ 4

During pregnancy, cigarettes/d

0

13 ⫾ 2

10 ⫾ 2

...........................................................................................................................................................................................................................

0

...........................................................................................................................................................................................................................

Smoking cessation, wk

...

6⫾1

...

.....................................................................................................................................................................................................................................

Parity

1.9 ⫾ 0.2

2.3 ⫾ 0.4

1.3 ⫾ 0.2

.....................................................................................................................................................................................................................................

Schooling, y

16 ⫾ 0

14 ⫾ 1

15 ⫾ 1

..............................................................................................................................................................................................................................................

Fetal characteristics

.....................................................................................................................................................................................................................................

Gestational age, d

273 ⫾ 1

269 ⫾ 2

274 ⫾ 2

3685 ⫾ 117

3202 ⫾ 122

3512 ⫾ 106

..................................................................................................................................................................................................................................... c

Birthweight, g

..................................................................................................................................................................................................................................... c c

Length, cm

52.1 ⫾ 0.6

49.7 ⫾ 0.5

52.0 ⫾ 0.6

Head circumference, cm

35.9 ⫾ 0.4

34.5 ⫾ 0.4

35.0 ⫾ 0.4

..................................................................................................................................................................................................................................... .....................................................................................................................................................................................................................................

Placental weight, g

727 ⫾ 39

707 ⫾ 43

698 ⫾ 54

.....................................................................................................................................................................................................................................

5-min Apgar score

10

Male sex, n (%)

10 (67)

10

10

.....................................................................................................................................................................................................................................

3 (30)

2 (22)

7.29 ⫾ 0.01

7.31 ⫾ 0.02

.....................................................................................................................................................................................................................................

pH of cord blood

7.30 ⫾ 0.01

..............................................................................................................................................................................................................................................

Values are mean ⫾ SEM or no. (%). Continuous data were analyzed by Kruskal-Wallis test, followed by 3 pairwise comparisons of means with Bonferroni correction to account for multiple comparisons and categorical data by ␹2 test. BMI, body mass index before pregnancy; BP, blood pressure. a

Detection limit for cotinine is 10 ng/mL; b P ⬍ .005 compared to smokers; c P ⬍ .05 compared to smokers.

Andersen. Maternal smoking and uterine vasorelaxation. Am J Obstet Gynecol 2011.

detectable levels of cotinine, while 100% of the samples of smokers were cotinine-positive. The newborns of smokers had a lower weight and were shorter than newborns of nonsmokers and exsmokers (Table 1). However, no differences were found in the head circumference and placental weight of the 3 infant groups. The Apgar score at 5 minutes and the pH of the umbilical cord blood were similar for nonsmokers, smokers, and ex-smokers. Surprisingly, only 30% of the infants among smokers and 22% among ex-smokers were male, compared to 67% among nonsmokers. However, no statistical 177.e4

differences were found in infant sex ratios between the groups.

Vasoconstrictor response The tension development to U46619 was similar in uterine arteries obtained from nonsmokers, smokers, and ex-smokers (data not shown). Endothelium-dependent relaxation Figure 1 shows representative traces of wire myograph recordings of the isometric force of uterine small arteries from nonsmokers (top), smokers (middle), and a time control (bottom). The uterine small artery from the pregnant smoker demonstrates impaired endothelium-

American Journal of Obstetrics & Gynecology FEBRUARY 2011

dependent relaxation compared to the artery from the pregnant nonsmoker. The time control demonstrates no spontaneous relaxation. The concentration-dependent relaxation in response to bradykinin in the absence and presence of inhibitors in uterine arteries from nonsmokers, smokers, and ex-smokers is shown in Figure 2, while the AUC and Rmax are shown in Table 2. Bradykinin-induced relaxation, as measured by the AUC and Rmax, was significantly reduced in arteries of smokers and ex-smokers compared to arteries of nonsmokers (Figure 2, A). Indomethacin did not significantly affect the bradykinin-induced relaxation in arteries from any of the 3 groups. The significant differences in AUC and Rmax between groups were, however, almost abolished (Figure 2, B). In the presence of L-NNA, bradykinin-induced relaxation was completely abolished in arteries of smokers and ex-smokers, while arteries of nonsmokers relaxed significantly at high bradykinin concentrations with Rmax of ⬃40% (Figure 2, C). However, when both indomethacin and L-NNA were present, arteries from nonsmokers no longer responded to bradykinin (Figure 2, D). The bradykinin-induced relaxations were similar in arteries from nonsmoking women who delivered male infants (n ⫽ 7) compared to arteries from women who delivered female infants (n ⫽ 5) (data not shown).

Endothelium-independent relaxation The concentration-dependent relaxation to SNP is shown in Figure 3 and the AUC and Rmax are shown in Table 2. The relaxation to SNP in arteries of nonsmokers and smokers was similar but was decreased compared to arteries of ex-smokers as there was a smaller AUC compared to the arteries of nonsmokers.

C OMMENT The present study demonstrates that endothelium-dependent NO-mediated relaxation is impaired at full term in the uterine small arteries of pregnant smokers and in women who stop smoking early in pregnancy. In addition, the data show that endothelium-independent re-

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FIGURE 2

Concentration-dependent vasorelaxation in response to bradykinin

Concentration-dependent relaxation in response to bradykinin in uterine small arteries from nonsmokers (Œ), smokers (●), and ex-smokers (□) after incubation with physiological salt solution (A; n ⫽ 12, n ⫽ 9, and n ⫽ 5, respectively), 10 ␮mol/L indomethacin (INDO) (B; n ⫽ 12, n ⫽ 8, and n ⫽ 6), 100 ␮mol/L L-NG-nitroarginine (L-NNA) (C; n ⫽ 13, n ⫽ 8, and n ⫽ 5), or both INDO and L-NNA (D; n ⫽ 9, n ⫽ 7, and n ⫽ 5). Values are mean ⫾ SEM. Andersen. Maternal smoking and uterine vasorelaxation. Am J Obstet Gynecol 2011.

laxation in response to the NO donor SNP is better in the arteries of women who stop smoking early in pregnancy compared to those of nonsmoking and smoking women. In support of previous findings, we found that newborns of smokers were significantly smaller, as measured by weight and length, than newborns of

nonsmokers.1-6 We also found that women who stopped smoking early in pregnancy delivered infants that were not significantly smaller than those of nonsmokers.4-6 The infant sex ratios were, however, not ⬃50% among any of the 3 groups, and the smokers were not different with respect to years of schooling compared to the other 2 groups, as

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was expected.6,26,27 One plausible explanation for these findings is that the number of pregnant women included in the present study is low and that the power of the study to detect small differences in these variables is therefore low. In the present study, we found that endothelium-dependent bradykinin-induced relaxation was impaired in uterine small arteries obtained at term from smokers and ex-smokers. To determine the relative roles of NO, vasodilator prostanoid, and endothelium-derived hyperpolarizing factor (EDHF) in bradykinin-induced relaxation, inhibitors of eNOS and COX were used. In the presence of only the eNOS inhibitor L-NNA, the arteries from smokers and ex-smokers no longer responded to bradykinin although the arteries from nonsmokers relaxed at high bradykinin concentrations. In the presence of both L-NNA and the COX inhibitor indomethacin, the relaxation to bradykinin was completely abolished in all arteries. Our findings suggest that maternal smoking impairs endothelium-dependent NOmediated relaxation in uterine small arteries, and that this detrimental effect is not improved by smoking cessation early in pregnancy. However, it is possible that an abnormal prostanoid-mediated vasodilation also plays a role in the impaired endothelial function associated with smoking. The reported magnitude of EDHF responses in uterine arteries is variable. Mints et al28 found approximately 10% relaxation in arteries from nonpregnant women, while others found about 60-80% relaxation in arteries from pregnant women exposed to 1 ␮mol/L bradykinin in the presence of eNOS and COX inhibitors.29-31 In the present study, 0-8% relaxation was found. Although there is no explanation why such variability exists, our data suggest that an altered EDHF function does not explain the effect of smoking on endothelial function in uterine small arteries. The present findings are consistent with the known deleterious effects of smoking on endothelial function in nonpregnant individuals.15-17 Our finding that smoking cessation ⬃33 weeks before term did not abolish the effect on

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TABLE 2

FIGURE 3

Relaxation response of uterine small arteries from pregnant nonsmokers, smokers, and ex-smokers

Concentration-dependent vasorelaxation in response to sodium nitroprusside

Variable

Nonsmokers

Smokers

Ex-smokers

Bradykinin

(n ⫽ 12)

(n ⫽ 9)

(n ⫽ 5)

AUC

..................................................................................................................................................................................................................................... a b

314 ⫾ 14

412 ⫾ 18

399 ⫾ 20

(%)

93 ⫾ 2

62 ⫾ 10

79 ⫾ 11

Bradykinin after INDO

(n ⫽ 12)

(n ⫽ 8)

(n ⫽ 6)

349 ⫾ 20

441 ⫾ 18

389 ⫾ 28

..................................................................................................................................................................................................................................... b max ..............................................................................................................................................................................................................................................

R

..................................................................................................................................................................................................................................... b

AUC

.....................................................................................................................................................................................................................................

R

(%)

81 ⫾ 8

54 ⫾ 13

81 ⫾ 9

max ..............................................................................................................................................................................................................................................

Bradykinin after L-NNA

(n ⫽ 13)

(n ⫽ 8)

(n ⫽ 5)

472 ⫾ 11

501 ⫾ 8

503 ⫾ 7

.....................................................................................................................................................................................................................................

AUC

..................................................................................................................................................................................................................................... b b max ..............................................................................................................................................................................................................................................

R

(%)

Bradykinin after INDO and L-NNA

40 ⫾ 10

0.7 ⫾ 2

–1.9 ⫾ 4

(n ⫽ 9)

(n ⫽ 7)

(n ⫽ 5)

501 ⫾ 6

507 ⫾ 9

501 ⫾ 7

.....................................................................................................................................................................................................................................

AUC

.....................................................................................................................................................................................................................................

R

(%)

8.4 ⫾ 7

–1.4 ⫾ 2

1.6 ⫾ 2

max ..............................................................................................................................................................................................................................................

Sodium nitroprusside

(n ⫽ 11)

(n ⫽ 7)

(n ⫽ 7)

379 ⫾ 22

349 ⫾ 37

277 ⫾ 18

71 ⫾ 8

76 ⫾ 13

96 ⫾ 1

..................................................................................................................................................................................................................................... b

AUC

.....................................................................................................................................................................................................................................

Rmax (%)

..............................................................................................................................................................................................................................................

Values are mean ⫾ SEM. Data were analyzed by Kruskal-Wallis test, followed by 3 pairwise comparisons of means with Bonferroni correction to account for multiple comparisons. AUC, area under curve; INDO, indomethacin; L-NNA, L-NG-nitroarginine; Rmax, maximum relaxation. a

P ⬍ .005 compared to nonsmokers; b P ⬍ .05 compared to nonsmokers.

Andersen. Maternal smoking and uterine vasorelaxation. Am J Obstet Gynecol 2011.

endothelium-dependent NO-mediated relaxation in uterine arteries is in accordance with previous findings in nonpregnant individuals. Previous studies have shown that in healthy, young adults the endothelium-dependent vasodilation in the brachial artery was 2.5% in smokers, 5% ⬃6 years after smoking cessation, and 10% in nonsmokers.16 These findings suggest that the endothelial cells of ex-smokers slowly regain a function similar to that of nonsmokers. Interestingly, endothelial function as measured by eNOS activity and concentration in endothelial cells of umbilical cord and chorionic vessels obtained from women who quit smoking at ⬃6 weeks of gestation were similar to the levels in endothelial cells that had never been exposed.6 This suggests that fetal endothelial cells may rapidly regain normal function after exposure to smoking for a short period of time. Alternatively, the normally functioning endothelial cells of fetal vessels from women who stopped smoking early in pregnancy may have been 177.e6

formed after the women stopped smoking and were thus never exposed to smoke. In light of the data presented here, it would be interesting to quantify the level of eNOS activity and concentration in endothelial cells of maternal uterine arteries. Surprisingly, a recent study showed that endothelial-dependent bradykininmediated relaxation in myometrial small arteries isolated from women who smoked during their pregnancy was enhanced compared to women who were nonsmokers or ex-smokers.25 This finding contradicts data in nonpregnant individuals and our data presented here. Although the differences cannot be presently explained, they may be due to methodological differences between the studies. For instance, Myers et al25 used another PSS composition, induced vasoconstriction with arginine vasopressin (10 nmol/L), and included Asian and Afro-Caribbean women, who demonstrated diminished endothelial-dependent relaxation compared to Caucasian

American Journal of Obstetrics & Gynecology FEBRUARY 2011

Concentration-dependent relaxation in response to sodium nitroprusside in uterine small arteries from nonsmokers (Œ; n ⫽ 11), smokers (●; n ⫽ 7), and ex-smokers (□; n ⫽ 7). Values are mean ⫾ SEM. Andersen. Maternal smoking and uterine vasorelaxation. Am J Obstet Gynecol 2011.

women; however, no information was given on ethnicity and smoking habits. Intravenous infusion of nicotine reduces endothelium-dependent bradykinin-mediated vasodilation in the dorsal hand vein of healthy nonsmokers.18 Furthermore, a study on pregnant ewes found that endothelium-dependent NO-mediated relaxation was reduced in uterine arteries after 48 hours of incubation with nicotine.14 These observations support the hypothesis that nicotine acts directly on the endothelium to reduce NO production. The effect of smoking and smoking cessation on endothelium-independent relaxation can be assessed by exposure to the NO donor SNP. We found that SNPinduced relaxation was similar in arteries from nonsmokers and smokers.14 We also found that endothelium-independent relaxation was enhanced in uterine small arteries from women who stopped smoking early in pregnancy, which is consistent with a higher NO sensitivity of

www.AJOG.org the vascular smooth muscle in that group.32 However, if smooth muscle sensitivity to NO is higher in arteries of ex-smokers, we expected to find a better bradykinin-mediated relaxation. Perhaps this finding is related to the other surprising observation of this study that women who stopped smoking produced infants of a normal size yet have reduced vasodilator responses to bradykinin. One possible explanation for this is that the arteries from the isthmus studied here behave differently from those directly supplying the placenta. An alternative explanation is that smoking-associated changes in uterine vasorelaxation are of minor importance for fetal growth compared to the effect of smoking on the endothelial cells of the fetal vascular bed. In conclusion, the findings suggest that maternal smoking reduces endothelium-dependent NO-mediated relaxation in the uterine vascular bed, which by reduced uterine blood flow may contribute to restricted fetal growth. Additionally, the findings suggest that smoking cessation early in pregnancy does not fully abolish this deleterious effect on the uterine vessels, despite an improvement in smooth muscle sensitivity to nitroprusside. f ACKNOWLEDGMENTS We thank Associate Professor Tine Brink Henriksen (Department of Pediatrics, Aarhus University Hospital, Skejby) for access to data from the Aarhus Birth Cohort and Professor Michael Vaeth (Department of Biostatistics, Aarhus University) for statistical supervising. Furthermore, Kirsten Zeeberg (Department of Obstetric and Gynecology) and technicians (Department of Clinical Biochemistry) are acknowledged for their technical assistance. We thank the employees of the Department of Obstetrics and Gynecology for their cooperation in this study.

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