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Pregnancy |
a Perinatal Research Centre, Departments of Obstetrics/Gynecology and Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 252
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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nitric oxide, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal models examining vascular adaptations to pregnancy have been extensively studied. In particular, systemic blood vessels (small mesenteric arteries) from pregnant rats have been investigated. Similar to changes in vascular function observed in humans [2], rat pregnancy is associated with a blunted systemic vasoconstrictor response to adrenergic agonists [3] and angiotensin [4] as well as an enhanced endothelial-dependent vasodilation [5, 6]. The increase in endothelial-dependent vasodilation in the rat is mediated mainly by enhanced nitric oxide (NO) modulation of vascular tone [5]. Systemic vessels from pregnant rats may also be more sensitive to low doses of endothelial-independent vasodilators [6], suggesting enhanced smooth muscle sensitivity during pregnancy.
The prostaglandin H synthase (PGHS) pathway also regulates vascular reactivity via the production of vasoactive factors, including prostacyclin, a potent vasodilator. In pregnant rats, PGHS-dependent vasodilation does not seem to play an important role in the vascular adaptations [3], which is consistent with data illustrating that infusion of indomethacin (PGHS inhibitor) into pregnant rats does not induce hypertension [7]. However, a non-NO, nonprostacyclin-dependent hyperpolarizing factor may be involved in the systemic vascular adaptation to pregnancy in the rat [6].
The uterine circulation is also known to undergo dramatic alterations during pregnancy, leading to increased endothelial-dependent vasodilation. In the rat uterine artery, enhanced NO-mediated relaxation is an important adaptation during pregnancy [8], which is similar to mechanisms involved in sheep pregnancy [9]. However, studies that measure in vivo perfusion pressure of the rat uterine circulation suggest that endothelial-derived hyperpolarizing factors (EDHFs) are also involved [10]. The difference between these studies may be explained by the fact that the in vitro study in the uterine artery did not take into account the downstream small-caliber vessels, which are know to have greater EDHF-dependent vasorelaxation [11].
Mouse pregnancy may be a suitable model for the study of human pregnancy, considering the similarities in trophoblast invasion and placental development [12]. Furthermore, a recent study investigating cardiovascular changes in early and late mouse gestation found that mean arterial pressure was reduced in early pregnancy, while by late gestation, the cardiac output was increased and the pressor response angiotensin was reduced [13]. These data are in accordance with cardiovascular changes in human pregnancy [1]. However, the mechanisms responsible for these cardiovascular changes during pregnancy are not fully understood.
Recent advances in genetic manipulations have lead to the development of transgenic mouse models, many of which focus on elucidating the mechanisms involved in pregnancy adaptations. For instance, cardiovascular abnormalities have been documented during pregnancy in adrenomedullin knockout mice [14], in renin-angiotensin overexpressing mice [15], as well as in an inbred mildly hypertensive mouse strain (BPH/5) [16]. Yet these studies did not investigate the specific pathways mediating the cardiovascular changes. Based on the importance of the NO pathway in rat gestation, many thought that NO synthase knockout (NOS-/-) mice would provide a good model to study mechanisms of abnormal vascular adaptation to pregnancy. However, these mice do not become hypertensive and in fact appear to have a normal pregnancy [17]. Therefore, vascular adaptations in the mouse may involve different mechanisms than in the rat. However, few studies have been specifically designed to investigate the mechanisms mediating changes in vascular function during normal mouse pregnancy. We hypothesized that mouse pregnancy will involve enhanced vasodilation, mediated by an increase in both NOS- and PGHS-dependent pathways, as well as enhanced EDHF-like relaxation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female C57BL/6J mice (12 wk of age) were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in a temperature- and humidity-controlled environment under 12L:12D cycles. Water and standard laboratory rodent chow were available ad libitum to the mice. Females were bred for 2 h (1:2 male to female ratio), and the presence of a seminal plug signified successful mating (Day 0 of gestation). Experiments were performed on Day 17–18 of gestation; delivery occurs on Day 19 of gestation. Nonpregnant mice were used at various times throughout their estrous cycle. However, there was not a significant variation in vessel function among groups and therefore data were pooled. On the day of the experiment, mice were killed by cervical dislocation. These protocols were approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee and were in accordance with the Canadian Council on Animal Care.
Vessel Preparation and Wire Myography
The uterus and mesenteric arcade were removed from the animal and placed immediately into ice-cold Delbecco modified essential medium (DMEM) buffer (1 mM sodium pyruvate, 25 mM sodium bicarbonate, 5 mM Hepes, 5 mM D+ glucose), which was used to maintain vessel viability [18]. The uterine artery (100 and 200 µm; nonpregnant and pregnant, respectively) and second-order mesenteric arteries (150 µm) were cleaned free of fat and connective tissue under a light microscope. After threading with two smooth 20-µm tungsten wires, vessels were mounted in an isometric wire myograph system (Kent Scientific, Litchfield, CT), warmed to 37°C (bathed in 5 ml of DMEM buffer), and equilibrated for 30 min.
Experimental Protocol
We chose to study the uterine and mesenteric vasculature for distinct reasons. The uterine artery is known to undergo dramatic physiological changes and restructuring during pregnancy due to the large increase in blood flow to the placenta. On the other hand, small mesenteric blood vessels are important in regulating overall peripheral vascular resistance [19]. Because pregnancy is a state of generalized vasodilation and mesenteric blood flow is known to increase during gestation [20], it is important to understand how this vasculature has adapted to account for these changes. Furthermore, by comparing two vascular beds, we were able to elucidate whether different mechanisms are involved in the vascular changes. Our initial studies investigated vascular function in the aorta, and there were no differences in vascular function between pregnant and nonpregnant mice (data not shown).
At the start of each experiment, vessel length was measured using a micrometer and a passive circumference-tension curve was performed for each vessel to determine the optimum resting tension. Cumulative dose-response curves were initially performed for phenylephrine (10-6 to 10-4 M). Vessels were used for subsequent relaxation curves if they attained a minimum of 1.0 mN/mm of tension in response to phenylephrine. The concentration of phenylephrine required to produce an 80% response (EC80) was used to preconstrict the vessels. Methacholine (muscarinic-agonist; endothelial-dependent vasodilator) dose-response curves were performed alone or after a 15-min preincubation with L-NAME alone (NOS inhibitor; 100 µM); meclofenamate alone (MECLO; PGHS inhibitor; 10 µM); L-NAME and MECLO together; L-NAME, MECLO, plus inhibitors of calcium-sensitive potassium channels (apamine; 10 µM and charybdotoxin; 0.1 µM). Endothelial-independent relaxation was also assessed in response to sodium nitroprusside (SNP). All vasorelaxation dose-response curves were performed in the range of 10-8 to 10-5 M.
Data Analysis and Statistics
Dose-response curves are graphically depicted as percent relaxation, and each point represents mean ± standard error of the mean from at least seven separate animals. Prior to the use of inhibitors, the slopes of the dose-response curves were similar between groups. Therefore, EC50 concentrations were calculated for methacholine-induced and SNP-induced relaxation, and these values were compared between nonpregnant and pregnant mice using a Mann-Whitney rank sum test. The effect of L-NAME and/or MECLO on methacholine-induced dilation was analyzed by comparing maximum relaxation to methacholine in the presence or absence of inhibitors. Maximum relaxation was used to compare groups due to the large reduction in relaxation in the presence of the drugs (especially in the vessels from pregnant mice); thus, the EC50 values were not applicable. Statistical significance was determined using a one-way ANOVA on ranks (due to small numbers in the data set). P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-mediated vasoconstriction using phenylephrine. Uterine and mesenteric vessels from pregnant mice had a blunted sensitivity to phenylephrine compared with nonpregnant mice but only at the low range of doses (data not shown). However, EC80 concentrations were not different between groups (uterine EC80: 5.6 ± 0.96 µM versus 7.7 ± 0.9 µM; P = 0.2; mesenteric EC80: 8.3 ± 2.2 µM versus 11 ± 1.2 µM, P = 0.09). For this reason, we used the EC80 dose to preconstrict vessels for subsequent relaxation curves. Furthermore, preconstricting vessels with the EC80 concentration provided a greater range of tensions over which relaxation could be measured. As hypothesized, methacholine-induced relaxation was significantly enhanced during pregnancy in both uterine (Fig. 1A) and mesenteric blood vessels (Fig. 1B); however, the effect was more pronounced in the uterine vasculature. To address whether NOS and/or PGHS were involved in the enhanced vasodilation of pregnancy, methacholine-induced relaxation was assessed in the presence or absence of specific inhibitors to these pathways. In the nonpregnant uterine vasculature, preincubation with either L-NAME or MECLO did not alter relaxation to methacholine (Fig. 2A). However, in uterine arteries from pregnant mice, blocking either NOS or PGHS shifted the dose-response curve to the right, indicating an impaired relaxation capacity (Fig. 2B). By comparing the maximum relaxation to methacholine before and after inhibitors, it is evident that both L-NAME and MECLO significantly reduced relaxation in uterine arteries from pregnant mice (Fig. 2B, inset). These results suggest that both NOS- and PGHS-dependent relaxation is enhanced in the uterine artery from pregnant mice.
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Unlike the uterine vasculature, preincubation with L-NAME in nonpregnant mesenteric arteries impaired methacholine-induced relaxation, although the effect of MECLO treatment was minimal (Fig. 3A and inset). In mesenteric arteries from pregnant mice, both L-NAME and MECLO alone inhibited the relaxation to methacholine to a similar extent. Indeed, the maximum relaxation to methacholine was significantly reduced after blocking either NOS or PGHS (Fig. 3B, inset).
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Endothelial-dependent relaxation that is insensitive to NOS and PGHS antagonists is thought to be due to EDHF-like molecule(s) [11]. By preincubating the vessels with the combination of L-NAME and MECLO, we were able to examine the remaining EDHF-like relaxation to methacholine. Our data illustrate that both uterine and mesenteric arteries have non-NO, nonprostacyclin-mediated relaxation from both nonpregnant and pregnant mice. A comparison of the percent maximal relaxation to methacholine (in the presence of L-NAME and MECLO) shows that, in the uterine artery, the remaining methacholine-induced relaxation is reduced in pregnant mice (Fig. 4A). However, in mesenteric arteries, there is no difference in the relaxation between pregnant and nonpregnant vessels (Fig. 4B). Because hyperpolarizing factors act via potassium channels to induced relaxation, we preincubated vessels with a combination of potassium channel blockers (apamine and charybdotoxin) together with the NOS and PGHS inhibitors. In all vessel types from both nonpregnant and pregnant mice, relaxation to methacholine was completely abolished (data not shown), suggesting that this EDHF-like molecule is characteristically working through potassium channels to induce relaxation.
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We also assessed endothelial-independent vasorelaxation using SNP. Uterine vessels from pregnant mice were significantly more sensitive to SNP than vessels from nonpregnant mice, indicated by a left shift in the dose-response curve and a reduced EC50 concentration (Fig. 5A and inset). However, there was no difference in SNP-induced relaxation between nonpregnant and pregnant mesenteric vessels (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study of vessel function in mice is currently an emerging field. Many studies conducted on mouse vessel function have compared transgenic mice with their controls [21–24]. In our study using mesenteric arteries, the nonpregnant control mice exhibited both NO- and EDHF-like relaxation. In agreement with our data, mesenteric artery relaxation to acetylcholine was mainly NO dependent, in female control animals for transgenic mice overexpressing the growth hormone gene. However, the PGHS pathway was not investigated in this study [22]. In eNOS+/+ (wild-type) mice, blocking NOS, but not PGHS, partially inhibited acetylcholine-induced relaxation in mesenteric vessels [23], which is also in accordance with our results. However, the combination of NOS and PGHS inhibitors completely eliminated endothelial-dependent relaxation in eNOS+/+ mice [23], suggesting that EDHF does not play a significant role in mesenteric relaxation. In contrast, our data illustrates that, after inhibiting both NOS and PGHS in nonpregnant mice, approximately 35% of the methacholine-induced relaxation persists. The discrepancy between our study and that of Chataigneau et al. [23] may be due to strain variability, which can alter vascular responses [25]. However, it is also possible that differences are due to the fact that our experiments involved only female mice, while the data in the above study did not specify gender and likely included pooled data from both sexes. Indeed, gender can play a large role in many aspects of vascular function.
Interestingly, our results demonstrate that, in mesenteric arteries from pregnant mice, the effect of blocking NOS or PGHS alone was almost identical. This suggests that, during pregnancy, the NOS and PGHS pathways are redundant such that, when one is inhibited, the other provides adequate relaxation. Indeed, we also found that non-NO, nonprostacyclin-dependent relaxation is not significantly different between mesenteric arteries from nonpregnant and pregnant mice. Although this data suggests that hyperpolarizing factors are not involved in the mesenteric adaptations to normal mouse pregnancy, during pathological conditions, EDHF-like pathway(s) may be upregulated, thus compensating for a vasodilator that is lacking. In mice that lack eNOS, there is enhanced sensitivity to PGHS inhibitors compared with wild-type mice [23] as well as increased EDHF-mediated relaxation [23, 24]. These observations are in accordance with the above theory and may also explain why NOS-/- mice are not hypertensive during pregnancy, although this hypothesis has not been specifically investigated [17].
Uterine artery reactivity has not been studied in the mouse; however, pregnancy adaptations in the uterine artery of the rat and sheep are well characterized. Previous to the discovery of EDHF, studies in the rat showed that NO was responsible for the increase in vasodilation and reduced pressor response during pregnancy [8]. However, recent evidence suggests that an EDHF-like molecule plays a role in both rat uterine artery [10] and human myometrial vessel [26] relaxation during pregnancy. In sheep, enhanced uterine blood flow likely involves both NOS and PGHS pathways because NO and prostacyclin metabolites are elevated during ovine pregnancy [27, 28]. In the uterine artery from pregnant mice, we found that, in the presence of NOS and PGHS inhibitors, there was a significant reduction in relaxation, suggesting that these pathways have a role in the enhanced sensitivity to methacholine. In uterine vessels from nonpregnant mice, the effect of inhibiting NOS or PGHS was minimal. We also showed that the uterine artery was more sensitive to endothelial-independent vasodilators during pregnancy, suggesting increased smooth muscle sensitivity, possibly to NO. Finally, our data suggest that EDHF-like relaxation is reduced in uterine artery from pregnant mice. It is possible that enhanced relaxation mediated by the NOS and PGHS pathways is so great in the mouse that it minimizes contributions of hyperpolarizing factors to vascular adaptations to pregnancy in the uterine artery.
This study highlights the pathways involved in vascular adaptations during mouse pregnancy, which may be distinct from those in the rat. We found that although NO is involved in the vasodilation of mouse pregnancy, the PGHS pathway is also an important mediator of endothelial-dependent relaxation in both the uterine and mesenteric vasculature. Conversely, prostacyclin does not seem to play an important role in vascular adaptations to rat pregnancy. For example, corticotropin-releasing hormone-induced relaxation was not inhibited by blocking PGHS in pregnant rats, and the blunted pressor response to phenylephrine was prevented by preincubation with L-NAME but not meclofenamate [3, 29]. Thus, our data illustrate that vascular reactivity in mouse pregnancy is distinct from the rat. Furthermore, this study describes the mechanisms involved in both uterine and mesenteric adaptations to pregnancy, information that is necessary to interpret and understand altered vascular function in transgenic mouse models of pregnancy.
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FOOTNOTES |
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2 Correspondence: Sandra T. Davidge, Perinatal Research Centre, 232 HMRC, Departments of Ob/Gyn and Physiology, University of Alberta, Edmonton, AB, Canada T6G 2S2. FAX: 780 492 1308; e-mail: sandra.davidge{at}ualberta.ca
Received: 13 August 2002.
First decision: 3 September 2002.
Accepted: 3 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 10 µM). A) Methacholine-induced dilation nonpregnant uterine arteries (•; n = 7) and B) methacholine-induced dilation in pregnant uterine arteries (;oc; n = 10). Responses are expressed as percent relaxation and each value represents the mean ± SEM. Inset: Bars represent maximum relaxation to methacholine (averaged for n = 7 or 10 experiments). *P < 0.05 versus maximum relaxation to methacholine alone
