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BOR - Papers in Press, published online ahead of print December 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.064899
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BIOLOGY OF REPRODUCTION 78, 744–751 (2008)
DOI: 10.1095/biolreprod.107.064899
© 2008 by the Society for the Study of Reproduction, Inc.

Pregnancy; 

Regulation of Maternal and Fetal Hemodynamics by Heme Oxygenase in Mice1

Hui Zhao 2 3, Ronald J. Wong 3, Timothy C. Doyle 3, Nihar Nayak 4, Hendrik J. Vreman 3, Christopher H. Contag 3, and David K. Stevenson 3

Departments of Pediatrics3 and Obstetrics and Gynecology,4 Stanford University School of Medicine, Stanford, California 94305

ABSTRACT

Heme oxygenase (HMOX) regulates vascular tone and blood pressure through the production of carbon monoxide (CO), a vasodilator derived from the heme degradation pathway. During pregnancy, the maternal circulation undergoes significant adaptations to accommodate the hemodynamic demands of the developing fetus. Our objective was to investigate the role of HMOX on maternal and fetal hemodynamics during pregnancy in a mouse model. We measured and compared maternal tissue and placental HMOX activity and endogenous CO production, represented by excreted CO and carboxyhemoglobin levels, during pregnancy (Embryonic Days 12.5–15.5) to nonpregnant controls. Micro-ultrasound was used to monitor maternal abdominal aorta diameters as well as blood flow velocities and diameters of fetal umbilical arteries. Tin mesoporphyrin, a potent HMOX inhibitor, was used to inhibit HMOX activity. Changes in maternal vascular tone were monitored by tail cuff blood pressure measurements. Effects of HMOX inhibition on placental structures were assessed by histology. We showed that maternal tissue and placental HMOX activity and CO production were significantly elevated during pregnancy. When HMOX in the placenta was inhibited, maternal and fetal hemodynamics underwent significant changes, with maternal blood pressures increasing. We concluded that increases in maternal tissue and placental HMOX activity contribute to the regulation of peripheral vascular resistance and therefore are important for the maintenance of normal maternal vascular tone and fetal hemodynamic functions during pregnancy.

carbon monoxide, heme oxygenase, hemodynamic function, hemodynamics, placenta, pregnancy

INTRODUCTION

During pregnancy, the maternal body undergoes significant hemodynamic changes to ensure normal fetal growth. On average, maternal cardiac output can increase up to 30%–45%, resulting mostly from the intensified metabolism, increased circulating blood mass, the appearance of an accessory placental circulatory system, and finally, a gradual increase in body weight during pregnancy. Although blood volume increases, the systemic blood pressure in a healthy mother undergoes no significant changes. This is mainly due to a decrease in total peripheral vascular resistance (PVR), primarily at the arteriolar level [1, 2]. The mammalian placenta is also important for the regulation of both the maternal and fetal circulations. Adequate uterine blood flow is critical to fetal growth and development. The persistence of an abnormally high uteroplacental resistance is a strong predisposing factor for intrauterine growth retardation and preeclampsia [3, 4].

Heme oxygenase (HMOX) is the rate-limiting enzyme in the heme degradation pathway, which produces equimolar quantities of carbon monoxide (CO), iron, and biliverdin. Biliverdin is rapidly converted to bilirubin by biliverdin reductase [5]. To date, two primary isoforms of HMOX have been identified in the human and rodent: the inducible HMOX1, also known as HSP32 or stress-responsive protein, and the constitutive HMOX2 [6–8]. Besides having roles in maintaining homeostasis through the regulation of cellular heme and hemoprotein levels [9, 10], HMOX isozymes also have vasodilatory, anti-inflammatory, antiapoptotic, and antioxidant properties—all mediated primarily through its bioactive products, CO [10–12] and bilirubin/biliverdin [13].

The relevance of HMOX to human pregnancy disorders has been reported. Down-modulation of placental HMOX expression was found to be associated with recurrent miscarriages, spontaneous abortions, and preeclampsia [14, 15]. Genetic variations in Hmox1 expression in humans with microsatellite polymorphisms has been linked to idiopathic recurrent miscarriages and susceptibilities to emphysema [16]. Production of CO by human umbilical cord tissues has been demonstrated [17], and end tidal breath CO levels were found to be significantly lower in women with pregnancy-induced hypertension compared to controls [18]. In experimental rodent models, Hmox1 was found to be highly expressed in placenta [19, 20]. Chronic inhibition of HMOX1 during gestational ages Embryonic Day (E) 5–E14 leads to massive fetal resorption with no live births [21]. Overexpression of Hmox1 by adenoviral transfer improves pregnancy outcomes in a mouse model of abortion [22]. All of these findings strongly suggest a role for HMOX and CO in the maintenance of a normal, healthy pregnancy.

Accumulating evidence suggests the important role of HMOX on regulating blood pressure, mediated primarily through the vasodilatory effect of CO [23, 24]. CO, like nitric oxide (NO), is a diffusible gaseous molecule involved in cell signaling in a variety of cellular functions, including vasodilation and smooth muscle relaxation [11, 12, 25, 26]. There are at least two mechanisms involved in CO-mediated vasodilation: 1) activation of a relaxant mechanism (soluble guanylyl cyclase-based) and 2) interference with a constrictor mechanism (cytochrome P450-based) [27–29]. Ex vivo evidence has revealed that CO dilates isolated porcine coronary arteries, rabbit aortas, and rat thoracic aortas. In vivo, acute or chronic administration of inducers or substrates of heme oxygenase decreases blood pressure in spontaneously hypertensive rats [30–33]. In addition, delivery of the human HMOX1 gene into rat kidneys decreases blood pressure in a spontaneously hypertensive rat model [34]. More recently, direct evidence in mouse studies shows that administration of inhaled CO reverses pulmonary arterial hypertension [35]. Administration of CO-releasing molecules results in a mild decrease in mean arterial pressures [36] and significant increases in renal blood flow [37]. However, the role of HMOX/CO on maternal vascular tone regulation during pregnancy is not quite understood.

In this study, we hypothesized that HMOX contributes to the maintenance of normal vascular tone through the local and systemic production of CO, which then leads to vascular dilation in tissues and a decrease in PVR during pregnancy. We speculate that a decrease in CO production via inhibition of HMOX activity induces the constriction of arterial and placental vessels, subsequently up-regulating maternal blood pressure, reducing placenta perfusion, and leading to insufficient nutrition and gas exchange at the maternofetal interface.

MATERIALS AND METHODS

Reagents

Tin mesoporphyrin (SnMP, Frontier Scientific, Logan UT) was dissolved with 60 µl 4 M Na3PO4. After the addition of saline, the pH was titrated to 7.4 with 1 M HCl under constant stirring. The volume was then adjusted with saline to yield a solution of 4.0 mM. Solutions were kept in the dark and used within 3 h of preparation.

Animals

FVB mice (6–8 wk old) were obtained from Charles River Laboratories (Wilmington, MA) and maintained and bred under strict adherence to Stanford University institutional guidelines. Mice were bred at 6–8 wk of age. Gestational ages were determined by visualizing the vaginal plug (E0.5 = vaginal plug day) and confirmed by measuring embryonic length using micro-ultrasound. To evaluate the effect of SnMP, pregnant mice (n = 4) at E12.5 were administered SnMP (30 µmol/kg) or vehicle alone (controls) intravenously (IV), and tissue HMOX activity was measured at 2 and 24 h after administration.

Quantitative RT-PCR Analysis

After the animals were killed, placentas were collected and quickly placed in RNAlater (Qiagen, Germany). Total RNA was extracted using the RNAeasy Mini Kit (Qiagen) following the manufacturer's instructions. Hmox1, Hmox2, and beta-actin (Actb) mRNA levels were quantified using a Quanti-Tect SYBR Green RT-PCR kit (Qiagen), performed in a Stratagene Mx3005P (Stratagene, La Jolla, CA), detail as described previously [38].

Western Blot Analysis

We boiled 100 µg of the sonicates for 10 min in protein loading buffer and electrophoresed them. Proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA) using a semidry transblotter. The membrane was probed with HMOX1 and HMOX2 polyclonal antibodies (1:1000; StressGen, Victoria, BC Canada) and protein levels quantitated by densitometry as previously described [38].

Total Body CO Excretion Rates

Pregnant (~E12.5–E14.5, n = 4); nonpregnant, age-matched control mice (6–8 wk old, n = 4); and pregnant mice (~E12.5–E14.5, n = 4) treated with SnMP (IV 30 µmol/kg) were weighed and placed in a polypropylene chamber (50 ml) supplied with CO-free air at a flow rate of approximately 40 ml/min. After a 30-min equilibration period, the CO in the chamber outlet gas was determined by gas chromatography as previously described [39, 40]. Total body CO excretion (VeCO) was then calculated as µl CO excreted/h/kg body weight.

Carboxyhemoglobin

For carboxyhemoglobin (COHb) and total hemoglobin determinations, blood (75 µl) was collected by retro-orbital bleeding from pregnant (~E12.5–E14.5, n = 4) or nonpregnant, age-matched control mice (6–8 wk old, n = 4). Blood was then placed into specially prepared, custom-made capillary tubes containing heparin and saponin at time of death. A steel rod was inserted, stoppers were placed at each end of the tubes, and the blood was hemolyzed by shaking back and forth of the steel rod. The samples were stored at 4°C. COHb and total hemoglobin levels were determined by gas chromatography, and the Drabkin cyanmethemoglobin method, respectively, as previously described [41].

Total Plasma Bilirubin

Approximately 200 µl of blood collected as described was placed into 1.5-ml CapiJet blood collection tubes (Terumo Medical, Somerset, NJ), centrifuged at 13 000 x g to separate the plasma, and then stored at –20°C for total plasma bilirubin analysis as described previously [42]. Total plasma bilirubin levels were measured using a UB Analyzer UA-1 (Arrows, Osaka, Japan) and expressed as mg/dl.

HMOX Activity

Tissue HMOX activity was determined by gas chromatography through measurements of CO as previously described [39, 43, 44]. Briefly, mouse maternal tissues and placentas (E12.5–E14.5) and tissue from age-matched, nonpregnant mice (6–8 wk old) were harvested in ice-cold phosphate buffer (pH 7.5). Tissue sonicates (10% w/v) were incubated with equal (20 µl) volumes of NADPH (4.5 µM) and methemalbumin (50 µM/11.2 µM) for 15 min at 37°C in 2 ml CO-purged, septum-sealed vials. For abdominal aorta (AA) HMOX activity measurements, AAs from 2–3 mice were pooled for each sample, and tissue sonicates (2.5% w/v) were incubated with NADPH and methemalbumin for 30 min at 37°C. The amount of CO in the vial headspace was determined by gas chromatography. HMOX activity was expressed as nmol CO/h/g fresh weight (FW). In some cases, total organ HMOX capacity (nmol CO/h) was calculated by multiplying HMOX activity (nmol CO/h/g FW) by the FW of each organ (g).

Micro-Ultrasound

Umbilical artery blood flows, AA diameter, and umbilical artery diameter were detected using a Vevo 770 High-Resolution In Vivo Micro-Imaging System (VisualSonics, Toronto, Canada) fitted with a 40-MHz transducer. Mice were anesthetized with 5% isoflurane in 1 L O2/min and maintained anesthetized at 3% isoflurane in 1 L O2/min for the duration of the scan. Mice were kept warm on a heated platform (37°C) that was connected to a temperature/physiology monitor (Indus Instrument, Houston TX). Fur from the abdominal region was removed using a commercial hair remover (Nair; Church & Dwight Co., Inc., Princeton, NJ) and Ultrasound Transmission Gel (Parker Laboratories, Fairfield, NJ) was applied to this area. The B-mode was used to identify placentas, embryos, and maternal AAs. The power-Doppler mode was used to monitor blood flow velocities and pulse rates. Images were saved as static images or cine loops. During the entire procedure, physiological parameters such as heart rates and rhythms, respirations, and body temperatures were recorded. Images were annotated and quantified using an expansive library of preset measurements and software measurement tools.

Blood Pressure Monitoring

New sets of mice, pregnant (E12.5–E15.5, n = 4) or nonpregnant (6–8 wk old, n = 4), were treated with SnMP or vehicle controls and used for blood pressure measurement. Mice were anesthetized with sodium pentobarbital (Nembutal, 50 mg/kg; Abbott Laboratories, North Chicago, IL) given intraperitoneally and placed onto a warm plate. After 10–15 min, blood pressures were recorded using a rodent tail cuff blood pressure measuring system (MK-2000A, Muromachi Kikai, Tokyo, Japan). More than five measurements were taken for each mouse at 1–2 min intervals.

Histological Staining

Placentas (at ~E13) were dissected and collected in Fisher PROTOCOL* 10% Neutral Buffered Formalin (Fisher Scientific) for 24 h. The fixed placentas were embedded in paraffin according to standard protocol. We sectioned 6-µm-thick tissues from paraffin-embedded blocks using a microtome. Following deparaffinization, sections were stained by hematoxylin and eosin (H&E, American Master*Tech Scientific, Lodi, CA), and placenta structures were observed using a light microscope (Carl Zeiss Microimaging, Thornwood, NY).

Statistical Analysis

For comparison of experimental groups, paired or unpaired t-tests were performed for each set of experiments to determine whether statistically significant differences within and between the experimental groups existed at P ≤ 0.05.

RESULTS

Maternal Tissue and Placental HMOX Activity During Pregnancy

When we measured and compared HMOX activity in different tissues between age-matched pregnant (E12.5–E15.5) and nonpregnant mice, we found that HMOX activity (nmol CO/h/g FW) was significantly higher in maternal liver (by 39%, P < 0.05), spleen (by 16%, P < 0.001), and kidney (by 8%, P < 0.05). We also observed significant increases in FWs of maternal livers (by >60%, P < 0.05) and spleens (by >50%, P < 0.05). Therefore, the total organ HMOX capacity (nmol/h), calculated by multiplying tissue HMOX activity (nmol CO/h/g FW) to tissue weight (g), was also significantly elevated in maternal tissues (Table 1).


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  		TABLE 1. HMOX activity in tissues from nonpregnant (controls) and pregnant mice (E13.5–E14.5) and placentas at various gestational ages.

For comparisons between HMOX activity in the AAs of pregnant and nonpregnant mice, we found a significant increase in HMOX activity (by ~13%, P < 0.05) during pregnancy (Table 1).

In addition, during E12.5–E15.5, the placenta, the organ unique to pregnancy, also contained very high levels of HMOX activity (Table 1). In fact, HMOX activity in the E14.5 placenta was comparable to that of the spleen (409 ± 6 vs. 439 ± 4 nmol CO/h/g FW, respectively). Total placental HMOX capacity was the second highest among all tissues studied. Both HMOX activity and total capacity reached peak levels around E14.5 (Table 1). This developmental profile of HMOX activity also correlated with Hmox1 and Hmox2 mRNA and HMOX1 and HMOX2 protein levels as measured by quantitative RT-PCR and Western blot, respectively (Fig. 1, A and B).


Figure 01
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  		FIG. 1. Developmental profiles of Hmox isozyme expression and endogenous CO production during pregnancy. Levels of Hmox1 (black) and Hmox2 (red) isozymes in mRNA (A) and protein (B) were measured in placentas harvested from pregnant mice at E11.5–E18.5 (n = 3 at each age). The values represented are relative levels to those at E11.5. VeCO (C; n = 6), COHb (D; n = 4), and total plasma bilirubin (E; n = 4) levels were measured in pregnant (E13.5 ± 1, black bars) and age-matched (6–8 wk old) nonpregnant (control, gray bars) mice. All data shown as mean ± SD. *P < 0.05, **P ≤ 0.01.

Increase of Endogenous CO Production During Pregnancy

Because HMOX catalyzes the degradation of heme to produce endogenous CO, body CO production, represented as total body CO excretion rates (VeCO) and blood COHb levels, was also measured during pregnancy. Compared to nonpregnant FVB female mice, pregnant mice at E12.5–E15.5 had higher VeCO (by 20%, P = 0.01) and COHb levels (by >80%, P < 0.01) (Fig. 1, C and D). Furthermore, total plasma bilirubin levels were also higher by 22% (P < 0.05) in pregnant mice (Fig. 1E).

Maternal Hemodynamic Changes During Pregnancy

To investigate whether the changes in HMOX expression are associated with changes in maternal hemodynamics during pregnancy, we monitored heart rates, respiratory rates, and blood pressures in pregnant mice at E12.5–E15.5. Compared to nonpregnant mice, there were minimal changes in heart rates and respiratory rates (data not shown), and systolic blood pressures were even lower (P < 0.001) (Table 2) in the pregnant animals. Maternal AAs were found to be significantly dilated (0.83 ± 0.04 mm) compared to nonpregnant control mice (0.68 ± 0.07 mm, P < 0.001) (Fig. 2, A and B).


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  		TABLE 2. Effect of HMOX inhibition on maternal blood pressure (mmHg).a


Figure 02
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  		FIG. 2. Maternal and fetal hemodynamic changes during pregnancy. A) Representative micro-ultrasound image showing the maternal AA. B) AA diameters (mm, mean ± SD) measured from pregnant (E13.5 ± 1, black bar) and age-matched nonpregnant mice (n = 5 for each age, gray bar). C) Representative micro-ultrasound image showing the placenta, umbilical cord, and fetal heart. D) Pulse wave image taken from the umbilical artery as captured in the Doppler mode and used to quantify the velocity of umbilical artery blood flows. E) Developmental profile of umbilical artery blood flow velocities (mm/sec, mean ± SD) at E9.5–E15.5. Each symbol represents an individual fetal umbilical artery velocity measurement at each gestational age. *P < 0.001.

Fetal Umbilical Artery Blood Flow Velocity

Fetal hemodynamic parameters were then measured using micro-ultrasound. A static image and a Doppler readout of umbilical artery blood flow velocities taken in utero of an E11.5 fetus are shown in Figure 2, C and D. Umbilical artery blood flow velocities and fetal heart rates were quantified from pulse waves obtained by Doppler readouts in pregnant mice at E9.5–E15.5. At E9.5, blood flow velocities were barely detectable, but from age E10.5–E12.5, blood flow velocities increased to 48–56 mm/sec (Fig. 2E). A significant increase was found at gestational age E13.5, which was 50% higher than that observed at E12.5. The upward trend continued until E14.5–E15.5.

Inhibition of HMOX Activity and CO Production by SnMP

To investigate the acute effects of HMOX deficiency on placental and fetal development, we first determined whether the administration of the potent competitive HMOX inhibitor, SnMP, could effectively reduce HMOX activity in the placenta and/or fetus. SnMP or vehicle alone (sham control) was administered IV to pregnant mice (E13.5 ± 0.5). At 2 and 24 h after treatment, placentas as well as maternal and fetal livers were collected, and HMOX activity was measured. At 2 h after treatment, significant reductions in HMOX activity were found in the placentas (to 10%, P < 0.005) and maternal livers (to 20%, P < 0.005) but there was only a minor inhibition in the fetal livers (to 86%, P < 0.005). Inhibition lasted for at least 24 h for all tissues studied—to 30% and 42% in placentas and maternal livers, respectively (Fig. 3A). Similarly, VeCO levels also were significantly decreased at 2 and 6 h post-SnMP administration (to 65% and 62%, respectively, P < 0.005) but returned to almost normal levels (to 90%) by 24 h (Fig. 3B). These data suggest that SnMP could effectively inhibit HMOX activity in the placenta while inhibiting HMOX activity only partially in the fetus.


Figure 03
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  		FIG. 3. Effect of inhibition of HMOX activity and CO production. A) HMOX activities in placentas and maternal livers were significantly inhibited by SnMP (30 µmol/kg body weight) at 2 and 24 h after SnMP administration (n = 4 for each time point) but were only slightly inhibited in the fetal liver. Ctrl, Control. B) VeCO rates were also significantly reduced at 2 and 6 h after SnMP administration (n = 4 for each time point). All data shown as mean ± SD. *P < 0.005.

Effect of HMOX Inhibition on Maternal Blood Pressure

To study the effect of HMOX inhibition on maternal vascular tone, we measured and compared blood pressures of pregnant mice (E12.5–E14.5) before and at 2 h after SnMP treatment. Significant increases in systolic (to 123%, P < 0.001), mean arterial (to 133%, P < 0.001), and diastolic (to 141%, P < 0.01) blood pressures were found at 2 h after SnMP treatment compared to baseline levels (Table 2). As expected, no increases were found in untreated pregnant mice. Interestingly, SnMP treatment resulted in no significant changes in blood pressures of nonpregnant mice and pregnant mice at early gestational ages (≤E10.5, data not shown).

Effect of HMOX Inhibition on Maternal AA Diameters

At 2 h following SnMP administration, dilated maternal AAs (0.86 ± 0.07 mm) were significantly constricted (0.56 ± 0.08 mm, P < 0.0001), and, after 24 h, they returned to levels (0.71 ± 0.03 mm) comparable to those of nonpregnant female mice (0.68 ± 0.07 mm, Fig. 4A). In addition, SnMP had no effect on AA diameters of nonpregnant mice.


Figure 04
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  		FIG. 4. Effect of SnMP on maternal and fetal hemodynamic function. Treatment with SnMP: (A) significantly constricted maternal AA diameters (n = 4 at each time point, mm, mean ± SD) during pregnancy but did not affect AAs of nonpregnant (control) mice (n = 4, at each time point); (B) had no effect on umbilical artery diameters (mm, mean ± SD); and (C) increased fetal umbilical artery velocities (% of control, mean ± SD) after 2 h (n = 4) but not after 24 h (n = 4). *P < 0.01; **P < 0.001.

Effect of HMOX Inhibition on Fetal Umbilical Artery Blood Flow Velocity

To study how decreases in maternal HMOX activity affect fetal cardiovascular function, we determined umbilical artery blood flow velocities and diameters pre- and post-SnMP treatment of pregnant mice at ~E12.5. Although umbilical artery diameters did not significantly change (Fig. 4B), blood flow velocities significantly increased 147% ± 11% at 2 h post-SnMP administration, compared to pregnant mice treated with vehicle (84 ± 9 vs. 61 ± 2 mm/sec, P < 0.01) (Fig. 4C). Overall increases in blood flow velocities were diminished at 24 h post-SnMP.

Effect of HMOX Inhibition on Placental Structure

Because placental HMOX activity was significantly inhibited by SnMP (Fig. 3A), we investigated whether any of the observed acute changes in hemodynamic function resulted from a disruption of placental structure. Placentas at ~E13 were collected, sectioned, and then stained with H&E. No major disruptions of placental structures were found at 2 h after SnMP treatment (Fig. 5, A and B). However, in the labyrinth areas, there was less maternal blood (nonnucleated cells) in the maternal vascular compartment and more fetal blood (nucleated cells) in the fetal capillary space, compared to nontreated control placentas (Fig. 5, C and D).


Figure 05
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  		FIG. 5. Effect of HMOX inhibition on placental histological structure. H&E-stained sections of placentas taken from an untreated (~E13.5), age-matched pregnant mouse (A and C) and from a mouse treated with SnMP (B and D) at E13.5. No major disruptions of placental structures can be seen in A and B (x100). Panels C and D (x400) show that in the labyrinth areas, there is less maternal blood (nonnucleated) in the maternal vascular compartment (yellow arrows) and more fetal blood (nucleated) in the fetal capillary space (black arrows) in an SnMP-treated placenta. l, Labyrinth; jz, junction zone. Bars = 50 µm (A, B); 20 µm (C, D).

DISCUSSION

Understanding the mechanism of hemodynamic regulation during pregnancy is fundamental to the development of therapeutic strategies for treating pregnancy disorders, such as pregnancy-induced hypertension, intrauterine growth retardation, and preeclampsia. In this study, we characterized the expression of Hmox in maternal tissues and the placenta and explored possible roles for HMOX/CO on maternal and fetal hemodynamic function in the mouse. We found significant increases in HMOX activity in major maternal organs such as liver, spleen, and heart (Table 1). We believe that the increase of HMOX activity in maternal organs is an adaptive mechanism for coping with the physiological challenges of pregnancy. An augmentation in circulating blood mass and erythropoiesis during pregnancy may lead to an increase in red blood cell turnover, subsequently leading to an increased heme load, which, in turn, induces Hmox1 expression. The observed enlargement of the liver and spleen may be the body's response to this increased demand.

During pregnancy, we observed significant dilation of AAs (Fig. 2, A and B) and no changes in blood pressure (Table 2) compared to nonpregnant mice. Dilation of AAs during pregnancy could result from an increase in AA HMOX activity (Table 1), leading to subsequent production of local CO levels. This speculation is supported by a previous study by Sammut et al. [45], which showed that CO produced from isolated rat aortic rings is a major contributor to the regulation of vascular tone. However, due to the fact that CO is diffusible and exists in circulation (as COHb), elevation of circulating CO levels (Fig. 1) produced from remote maternal tissues such as liver, spleen, and placenta could also execute a vasodilatory effect systemically. Even exogenous CO administration (CO inhalation and CO-releasing molecule) has been shown to regulate vascular tone in vivo [35, 36, 46]. The exact mechanism that contributes to the observed dilation of the AA is beyond the scope of this manuscript.

We also detected a very high level of HMOX expression (Fig. 1) and activity in the placenta—levels comparable to that of the normal (nonpregnant) spleen (Table 1), a tissue known for its high HMOX activity. Placental HMOX expression and activity peaked between E12.5 and E15.5, when placental vasculature is undergoing dramatic expansion and maturation. During this period, maternal blood pressures were not different from that in nonpregnant mice. When placental HMOX activity was inhibited, maternal blood pressures significantly increased (Table 2). In contrast, the same treatment did not affect blood pressures of nonpregnant mice or at earlier times in pregnancy (before E11.5), even though major maternal organs were also significantly inhibited by SnMP (data not shown). We speculated that reduction of placental CO production would lead to a constriction of local vessels and therefore an increase in placental PVR. Although we were unable to measure placental PVR noninvasively in vivo due to technical limitations, our hypothesis is supported by previous reports. Ahmed et al. [47], using isolated human placentas, showed that hemin-induced heme oxygenase-1 up-regulation could reduce vascular tension by 61% in preconstricted placental arteries. Lyall et al. [48] reported that resistance in a perfused placenta increases in a dose-dependent manner following inhibition of HMOX activity [48]. These data suggest that placental HMOX might be involved in the regulation of placental PVR, likely through the production of CO and a subsequent decrease in maternal vascular tone.

Up-regulation of umbilical artery velocity following SnMP administration (Fig. 4C) is not likely due to a direct effect on the fetal circulation. When SnMP was administered to the mother, we found only a small degree of HMOX inhibition (by 16%) in the fetal liver (Fig. 3A), suggesting that only a very limited amount of the drug was transported through the maternofetal blood barrier. We also found no changes in umbilical artery diameters post-SnMP treatment (Fig. 4B). In addition, according to our histological findings, there was a decrease in maternal blood cells in the maternal vascular compartment and an increase of fetal blood cells in the fetal capillary space following inhibition of HMOX activity (Fig. 5). These observations suggest that the fetal circulation increases in order to compensate for the poor maternofetal exchange in the placenta induced by maternal vessel constriction from the inhibition of HMOX.

In this study, we detected no detrimental effects of a single dose of SnMP on pregnancy outcomes. Actually, most of the parameters measured (such as VeCO, AA diameters, and fetal umbilical artery velocities) returned to normal (or at least near-normal) levels within 24 h post-SnMP administration (Figs. 3B and 4, A and B). Similar observations were also reported by Johnson et al. [49], who demonstrated that exogenous administration of CO only acutely lowers blood pressure in hypertensive rats. Inhibition of HMOX activity could result in the gradual accumulation of heme, which subsequently induces the expression of Hmox1, and, in turn, accelerates heme degradation and hence increases CO production. In fact, we have observed significant increases in HMOX1 protein within 24 h after SnMP administration [39]. In addition, changes in vascular tone following HMOX inhibition could be compensated for by other regulators, such as the renal angiotensin II system and NO [23]. Nitric oxide has a reciprocal relationship with CO. Ishikawa et al. [50] have shown that inhibition of CO production using a heme oxygenase inhibitor could augment regional NO formation in the rat pial microcirculation. It is interesting to note that nitric oxide synthase (NOS) levels are also elevated during pregnancy, and its inhibition can induce preeclampsia-like symptoms. More and more evidence has emerged, which reveals that there is a functional interaction between the heme-HMOX and the L-arginine-NOS systems. For example, NO interferes with the ability of CO to stimulate KCa channels in vascular smooth muscle cells, induces Hmox1 expression, and decreases HMOX activity as well as the activity of ferrochelatase, the terminal enzyme of the heme synthetic pathway. At the same time, CO attenuates NO-induced activation of soluble guanylyl cyclase and inhibits NOS [23, 51]. The fact that CO and NO affect each other's formation and action(s) raises the possibility that the vasoregulatory functions of the heme-HMOX and the L-arginine-NOS systems are intertwined, and the exact mechanism warrants further investigation.

Regulation of maternal vascular tone is a very complex process, contributed to and orchestrated by many factors [52]. That may explain the fact that the pathological mechanisms of pregnancy-induced hypertension and preeclampsia are still far from being understood despite tremendous efforts and intensive studies in this field. Our data suggest that the HMOX/CO system may be an important component in the regulation of maternal vascular tone. Besides its vasodilatory effect through CO, HMOX has antioxidative and cytoprotective properties that help to maintain the integrity of the vasculature wall and protect endothelial cells from oxidative stress. Therefore, investigating the roles of HMOX on vascular function during pregnancy will provide insight and deepen our understanding of the complexities of vascular tone regulation and have significant implications for further developing treatment strategies for complications of pregnancy.

ACKNOWLEDGMENTS

We thank Mrs. Peggy Kemper, Mrs. Pauline Chu, Mrs. Flora Kalish, and Drs. Stacy Burns-Guydish and Takashi Morisawa for their technical help. We also thank Drs. Richard D. Bland, Lucia M. Mokres, and Yuri Knauer for their assistance with blood pressure measurements.

FOOTNOTES

1Supported by National Institutes of Health grant HL58013, the Hess Research Fund, and the Mary L. Johnson Research Fund. Back

Correspondence: 2Hui Zhao, Department of Pediatrics, Division of Neonatology & Developmental Medicine, Stanford University School of Medicine, Grant Building S230, 300 Pasteur Drive, Stanford, CA 94305-5208. FAX: 650 725 7724; e-mail: huizhao2{at}stanford.edu

Received: 16 August 2007.

First decision: 6 September 2007.

Accepted: 20 November 2007.

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