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Ann Thorac Surg 2007;84:917-925
© 2007 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Role of Nitric Oxide Pathway in Placental Dysfunction Following Fetal Bypass

^a Division of Cardiothoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
^b Department of Obstetrics and Gynecology, University of Cincinnati, Cincinnati, Ohio

Accepted for publication April 16, 2007.

^_* Address correspondence to Dr Eghtesady, Division of Cardiothoracic Surgery, Cincinnati Children’s Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229-3039 (Email: pirooz.eghtesady{at}cchmc.org).

Presented at the Forty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 29–31, 2007.

	Abstract

Top Abstract Introduction Material and Methods Results Comment Discussion Acknowledgments References

 
Background: The etiology of placental dysfunction after fetal cardiopulmonary bypass remains unknown. The placental nitric oxide (NO) pathway has been implicated in this pathophysiology. We set out to examine possible perturbations in this pathway in an ovine model of fetal bypass.

Methods: Ovine fetuses (n = 14) between 100 and 114 days of gestation, instrumented to measure hemodynamics and umbilical blood flow, were placed on bypass for 30 minutes and followed after bypass for 2 hours. Sham controls (n = 6) were instrumented but did not undergo bypass. Real-time, in-vivo NO concentrations were measured in the placental circulation. To examine other components of the NO pathway, fetal plasma samples were analyzed by immunoassays for total NO metabolite and cyclic guanosine 3',5'–cyclic monophosphate (cGMP) levels. In addition, the expression of phosphodiesterase-5 was examined in placenta by immunohistochemistry. Statistical analysis was performed using analysis of variance with least significant difference post hoc tests (p 0.05).

Results: With the onset of bypass, an immediate increase occurs in umbilical NO concentrations. These return to baseline with cessation of bypass, and decline thereafter. In contrast, there was a linear increase in fetal plasma cGMP levels and a decline in NO metabolite concentrations through the post-bypass period. There was a dramatic increase in placental phosphodiesterase-5 expression with 30 minutes of bypass. The changes occur simultaneously with decreasing umbilical flows, increased placental vascular resistance, and worsening placental gas exchange.

Conclusions: Fetal bypass leads to significant reductions in placental NO concentrations despite increases in fetal plasma cGMP and placental phosphodiesterase-5 levels, indicative of perturbations in the fetal-placental NO pathway.

	Introduction

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Altered blood flow through the heart during intrauterine life is believed to be a major contributor in developing certain heart defects. The idea that restoration of normal flow in utero may prevent or change the evolution of these processes has led to increasing interest in maternal-fetal cardiac interventions. While great advances have been made using catheter-based interventions, progress in the application of open-heart surgery in the fetus remains limited [1–3].

Experimental studies since the 1980s have revealed that an increase in placental vascular resistance and deterioration of placental gas exchange invariably follows fetal bypass [4], manifested as progressive fetal hypercarbia, lactic acidosis, hypoxia, and finally fetal death [5].

The mechanism for this pathophysiology remains unknown, although several studies have implicated the nitric oxide (NO) pathway for the following reasons. First, the placenta is a rich source of NO that maintains this organ’s highly vasodilated state [6]. Second, pulsatile flow applied during fetal bypass improves fetal hemodynamics as well as decreases the degree and severity of the rise in placental vascular resistance [7, 8]. Pulsatile flow is known to stimulate NO synthesis and release by the vascular endothelium [4]. Third, administration of a specific inhibitor of NO synthesis eliminates the benefits of pulsatile fetal bypass [9]. Fourth, fetal gas exchange and placental vascular resistance after fetal bypass improve in the presence of a NO donor such as sodium nitroprusside [10]. Finally, the NO pathway has been shown to play a pivotal role in several pathophysiologic conditions affecting the placenta, such as preclampsia [11, 12].

The synthesis of NO from L-arginine, shown schematically in Figure 1, is dependent on activation of NO synthase [13], an isoform of which is located in the endothelial layer of blood vessels [14]. On diffusion to adjacent smooth muscle cells, NO activates soluble guanylate cyclase that stimulates the synthesis of cyclic guanosine 3',5'–cyclic monophosphate (cGMP). This initiates a sequence of protein phosphorylation reactions associated with smooth muscle relaxation, which in turn induces vasodilatation [15, 16]. The present study was undertaken to test the hypothesis that fetal bypass disrupts NO production in the fetal circulation and this contributes to the ensuing placental dysfunction. We examined this mechanism by measuring changes in the NO concentration of the placental circulation with fetal bypass using a novel method of continuous, direct measurement of endogenous NO and determining fetal plasma levels of cGMP and NO metabolites. Finally, we evaluated the expression of phosphodiesterase-5 (PDE-5), a key enzyme in the pathway responsible for regulating local cGMP concentrations.

View larger version (12K): [in this window] [in a new window]   Fig 1. Schematic representation of the nitric oxide–cyclic guanosine monophosphate (cGMP) signal transduction pathway leading to vasodilation. Nitric oxide (NO) synthesis from L-arginine is catalyzed by nitric oxide synthase (NOS). Rapid degradation of released NO produces citrulline and nitrates. Stimulation of guanylate cyclase activity by NO increases the production of cGMP. Degradation of cGMP is regulated by cGMP-specific phosphodiesterase-5 (PDE-5). Vasodilation is induced by increases in cGMP levels. (GTP = guanosine triphosphate.)

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment Discussion Acknowledgments References

Real-Time NO Sensor
Continuous measurements of NO concentrations in the fetal circulation were obtained by securing the tip of a 700-µm diameter probe (AmiNO-700; Innovative Instruments, Tampa, Florida) in the common umbilical vein. The NO concentration was continuously recorded throughout the procedure using a NO monitor, (inNO-T; Harvard Apparatus, Holliston, Massachusetts). The NO measurement system consists of an amperometric sensor, which utilizes the diffusion of NO through the membrane tip of the sensor from the sample solution. An electrical potential is then applied to the sensor element, which forces NO to lose an electron to the sensor element. A current is produced, in which the magnitude of the electrical current is proportional to the concentration of NO in the sample. The measurement is converted from picoAmps to nanoMolar concentration by the manufacturer’s supplied software inNO version 2.0. The sensor is calibrated before and after each experiment according to the manufacturer’s suggested method. Real-time NO measurements using similar NO sensors to the one used in the present study have previously been validated in other models [19, 20].

Fetal Bypass
Using methods previously described [17, 18], fetal bypass was performed after jugular vein cannulation using a 10F or 12F Bio-Medicus cannula (Medtronic, Minneapolis, Minnesota) and carotid artery cannulation using a 6F or 8F Bio-Medicus cannula. We intentionally avoided direct cannulation through sternotomy, to avoid the confounding effects of severe stress response from the sternotomy as reported previously [18, 21]. Based on our previous studies [17], we had a target flow rate of 200 to 250 mL · min^–1 · kg^–1. The pump system was normothermic and nonpulsatile, consisting of a roller pump with vacuum-assisted drainage and heat exchanger, and was primed with maternal donor blood [17, 22]. Bypass lasted for 30 minutes, and fetuses were then followed over the post-bypass period for 2 hours. Ewes and fetus were then euthanized for autopsy, measurement of fetal weight, and confirmation of catheter positions.

Sampling Regimen
Maternal and fetal arterial blood were collected before and after neck cannulation, at 15 and 30 minutes of bypass, and at 30, 60, 90, and 120 minutes after bypass. Blood gases were determined using an i-STAT clinical analyzer (i-STAT Corp, Windsor, New Jersey). Maternal and fetal lactate values were measured on an YSI 2300-STAT analyzer (YSI Corp, Yellow Springs, Ohio).

cGMP and NO Metabolite Immunoassays
Fetal blood samples for immunoassay were collected after neck cannulation at 30 minutes of bypass and at 30 and 120 minutes after bypass into lithium heparin-coated tubes (Monovettes; Sarstedt, Newton, North Carolina). The collected blood samples were immediately placed on ice and centrifuged, and the separated plasma was then frozen at –20°C until assay. The cGMP levels in fetal plasma were determined using a competitive enzyme-linked immunosorbent assay (ELISA) from Cayman Chemicals (Ann Arbor, Michigan) [23, 24]. In addition, NO metabolites (nitrites, or NO₂ ^–, and nitrates, or NO₃ ^–) levels in fetal plasma were measured using a colorimetric ELISA from Cayman Chemicals.

Immunohistochemistry
The placental expression of PDE-5 was determined after neck cannulation, and at 30 and 120 minutes after bypass using routine methods for tissue collection, localization, and specific primary antibodies to PDE-5, as previously described by our group [25]. Serial frozen sections (8 µm) cut from placentomes of experimental and control sheep were incubated with anti-human PDE-5 antibody (#4072; Cell Signaling Technology; available at: www.cellsignal.com) used at a dilution of 1:100. The sections were then incubated with biotin-labeled goat anti-rabbit immunoglobulin (Alexa-Fluor 568 #A11036 Molecular Probes; Invitrogen, Carlsbad, CA) used at 1:200 for 30 minutes. Negative controls were incubated with secondary antibody alone in the absence of primary antisera.

Statistical Analysis
To determine differences in measured parameters, the data were analyzed using type III analysis of variance (ANOVA) tests for between-group differences and least significant difference post hoc analysis for in-group differences, using a p value of 0.05 or less as statistically significant.

	Results

Top Abstract Introduction Material and Methods Results Comment Discussion Acknowledgments References

 
Arterial Blood Gases
The fetal values of arterial pH, pO₂, pCO₂, and hemoglobin saturation were similar during the pre bypass period in experimental and control animals (Fig 2). An overall comparison of fetal arterial blood gases between controls and bypass animals (treatment) indicated bypass significantly altered fetal pCO₂ (p = 0.017) and lactate (p = 0.001) as shown in Table 1. Post-hoc analysis of the changes in fetal arterial blood gases with bypass showed that at 30 minutes post-bypass fetal plasma concentrations of lactate (p = 0.019) and pCO₂ (p = 0.002) were significantly greater (Table 2, Fig 2A and B). In-group ANOVA analysis of the bypass values confirmed that this increase in pCO₂ was significant (p = 0.048). There were no significant changes in fetal pO₂ or oxygen saturation between experimental and control animals or with the conduct of bypass (Fig 2C and D, Tables 1 and 2). During fetal bypass, the physiologic fetal determinants of blood pressure (mean arterial pressure > 40 mm Hg), heart rate (150 ± 10 beats per minute), and umbilical flows (100 to 150 mL · kg^–1 · min^–1) were maintained.

View larger version (14K): [in this window] [in a new window]   Fig 2. Fetal arterial blood gases with cardiopulmonary bypass. Plasma concentrations of (A) lactate, (B) pCO2, (C) pH, and (D) pO2 and oxygen saturation (SO2) are shown for control animals (dashed lines) and experimental animals (solid lines). These variables were measured before bypass (pre Bypass), at 15 and 30 minutes of bypass, and at 30, 90, and 120 minutes after (Post) bypass. *p 0.05 comparing in-group versus prebypass (baseline). (In [D], solid lines with diamonds = bypass PO2; solid lines with triangles = bypass SO2; dashed lines with squares = control PO2; dashed lines with circles = control SO2.)

View this table: [in this window] [in a new window]   Table 2 Fetal Blood Gases Before, During, and After Bypass, and In-Group ANOVA With Least Significant Difference Post-Hoc Analysis Significance at p 0.05 Shown in Bold

View larger version (9K): [in this window] [in a new window]   Fig 3. Placental in-vivo concentrations of nitric oxide (NO) with cardiopulmonary bypass. The data are presented as percent changes in mean NO concentrations in experimental and control animals compared with values before bypass. In the bypass group (open bars [n = 7]), NO levels rose during the bypass period but gradually decreased after the bypass period, whereas the control group (solid bars [n = 6]) did not diverge from baseline.

View this table: [in this window] [in a new window]   Table 3 Placental In Vivo Nitric Oxide (NO) Concentration Before, During, and After Bypass and In-Group ANOVA With Least Significant Difference Post-Hoc Analysis Significance at p 0.05 Shown in Bold

View larger version (10K): [in this window] [in a new window]   Fig 4. Representative changes in-vivo nitric oxide (NO) concentrations (solid line) compared with umbilical blood flow (broken line) in an experimental fetus. Changes in umbilical blood flow (scale on left axis) paralleled changes in NO concentrations (scale on right axis), increasing during the 30-minute bypass procedure. Changes with cannulation before bypass (pre Bypass), and 30 and 60 minutes after bypass (post) until fetal demise are shown.

View this table: [in this window] [in a new window]   Table 4 Vasoactive Substances Release in Fetal Plasma Before, During, and After Bypass and In-Group ANOVA With Least Significant Difference Post-Hoc Analysis, Significance at p 0.05 shown in Bold

View larger version (18K): [in this window] [in a new window]   Fig 5. Changes in fetal plasma concentrations of (A) nitric oxide (NO) metabolites and (B) cyclic guanosine monophosphate (cGMP) concentrations with bypass expressed as percentage change from baseline (pre Bypass). (A) There is a significant and marked decline in the bypass group (open bars [n = 14]) in plasma NO metabolites concentrations at the end of 30 minutes of bypass, and 30 minutes and 120 minutes after bypass (post) when compared with baseline. (B) Plasma cGMP concentrations increased significantly in experimental animals (open bars), at 30 minutes and 120 minutes after bypass (post) when compared with baseline. *p 0.05 compared in-group with baseline. (Control = solid bars [n = 6].)

Placental Expression of PDE-5
To further examine potential mechanisms responsible for the rising cGMP levels, we assessed the expression of PDE-5, the key enzyme responsible for breakdown of cGMP, in placental tissue from experimental animals (Fig 6, panels A–C) and control animals (Fig 6, panels D–F) before bypass, and 30 minutes and 120 minutes after bypass. Unexpectedly, there was a dramatic increase in PDE-5 expression in the placenta at 30 minutes after bypass (Fig 6B, arrow). Maternal vascular smooth muscle cells, but not fetal cells of the placentome contained PDE-5 immunoreactivity, as previously reported by our group [25].

View larger version (48K): [in this window] [in a new window]   Fig 6. Representative pictures of phosphodiesterase-5 (PDE-5) expression in placental sections for (A, B, C) experimental animals and (D, E) control animals at x100 magnification detected by immunofluorescence. Immunohistochemical localization of PDE-5 is markedly increased 30 minutes after bypass (B, arrow) in tissues of maternal origin, but returned to baseline 120 minutes after bypass (C), similar to controls (D, E, F). Results were consistent in all animals.

	Comment

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In our study, we were able to detect a consistent surge in umbilical NO levels with the onset of bypass, which lasted during the period of extracorporeal support. Shortly after termination of bypass, however, the NO levels began a steep and persistent decline parallel with progressive deterioration in umbilical flows and placental gas exchange. We have no other study with which to compare our in-vivo NO data, although Vedrinne and colleagues [7] did measure NO metabolites after fetal bypass using pulsatile perfusion. They found plasma NO metabolite levels to be elevated with bypass in general (greater with pulsatile bypass), whereas we found the NO metabolite concentrations decreased approximately 50% from baseline. The Vedrinne study, however, was limited to measurements at a single time point during bypass (ie, no post-bypass period) and entailed a large volume of maternal blood prime (1 L) and use of an oxygenator. These differences may account for the different results between our studies. The increase in umbilical NO concentrations with bypass in our study would suggest a decrease in NO metabolism, as suggested by the reduction in measured NO metabolites.

Increases in NO with initiation of bypass suggest an activation of the placental endothelial cells leading to greater NO release. The increased NO levels can then, in turn, result in improvements in placental perfusion (and transient decrease in placental vascular resistance) as shown by parallel increases in measured umbilical blood flow. Shortly thereafter, however, the endothelial cells appear to lose the ability to sustain NO production. Simultaneously, there is a rise in fetal plasma lactate and pCO₂ consistent with worsening placental vascular resistance. This similar pattern of transitory compensatory increases in NO levels in the placental vasculature is seen at term in cases of placental insufficiency [30]. Among other causes, that may be due to disruption of the downstream NO signaling pathway. We therefore measured fetal plasma concentrations of the primary mediator of NO signaling in vasodilation, cGMP.

Surprisingly, we found a significant increase in cGMP concentration during the post-bypass recovery period but not during the conduct of bypass. An increase in fetal plasma cGMP levels with 30 minutes of bypass has previously been reported [7]; the post-bypass period, however, had not been previously evaluated. There can be several explanations for this paradoxic rise in cGMP levels. First, the increase may reflect the transient rise in placental NO levels with bypass. Alternatively, other pathways (eg, the natriuretic peptides) [31] that mediate actions through the cGMP second messenger signaling system could be activated with fetal bypass, leading to elevated cGMP levels despite declining NO concentrations. In addition, inactivation or inhibition of PDE-5, the enzyme that regulates degradation of cGMP in the uteroplacental vascular bed, could also lead to elevations in fetal plasma cGMP levels. For this latter reason, we examined placental expression of PDE-5.

An unexpected and marked increase in PDE-5 immunoreactivity was seen in the placenta of animals 30 minutes after exposure to extracorporeal circulation, although this increase was not sustained throughout the post-bypass period. We previously demonstrated the expression of PDE-5 in the ovine placenta [25] and noted that the majority of the expression of this protein occurs on the maternal side of the placental circulation. In view of our recent findings related to complex maternal-fetal interactions across the placental circulation [18], it is conceivable that placental PDE-5 could affect fetal cGMP concentrations. It is surprising, however, that we see elevated levels of cGMP in the setting of higher expression of the enzyme (PDE-5) that breaks down cGMP [32]. This effect is likely due to the PDE-5 gene promoter being positively regulated by cGMP [33]. Further studies are needed to determine whether increased expression of immunoreactive PDE-5 in fact reflects the active phosphorylated form of the enzyme. These findings suggest, however, that the elevated levels of cGMP are likely not reflective of the NO pathway, but rather arise from an alternative pathway or source. We are conducting further studies to examine the mechanisms that account for the observed changes and to further pinpoint the effects of fetal bypass on the placental NO pathway. These studies will include evaluations of the NO synthases that catalyze the synthesis of NO; expression of these enzymes has been shown to be altered in other placental pathophysiology such as preeclampsia [6].

In summary, we found fetal bypass induced significant perturbations of the fetal-placental NO signaling pathway. Disruption of the NO pathway is likely to contribute to the mechanisms mediating increased placental vascular resistance and placental dysfunction with fetal bypass.

	Discussion

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DR CHRISTOPHER A. CALDARONE (Toronto, Ontario, Canada): That’s very nice and elegant work. Could you speculate a bit about why you think the PDE-5 levels changed? Is this a change in RNA level transcription or increased degradation?

MR LAM: That is an excellent question. We don’t know if the change in PDE-5 expression is either the change in RNA levels or degradation. What we do know is the cells themselves are showing increased PDE-5 expression. So in future studies, we are examining how active the PDE-5 enzyme is itself. So we’re looking at phosphorylated levels of PDE-5, or if that the PDE-5 itself is being inhibited by some other substance to prevent it from breaking down the cGMP.

DR JOHN E. MAYER (Boston, MA): Do you know that the nitric oxide production is actually coming from the placenta itself, or could it be coming from white cells? And the second question related to that is, you obviously have looked at these samples histologically, and what can you tell us about the appearance of the microcirculation at least microscopically?

MR LAM: In some preliminary experiments we have seen similar observations regarding NO when using umbilical vein endothelial cells in an in vitro system, suggesting that the events indeed are occurring in the placenta and not secondary to other cells like passenger leukocytes/neutrophils in the microcirculation. Also, histologically what we see is that the endothelial cell barrier layer actually appears disrupted. We’re working on further clarifying these events by quantifying the e-NOS expression through Western blot and RT-PCR techniques.

DR MAYER: It may be coming from the placenta, but the question is, is there something lodging in the placenta that’s starting to make a lot of the so-called bad nitric oxide as opposed to the so-called good nitric oxide?

MR LAM: We don’t know for sure if it is coming from the e-NOS in placenta or if it’s coming from the i-NOS or inducible NOS, which I assume you are alluding to by bad nitric oxide, since it is the primary nitric oxide released in response to stress or inflammation.

DR MAYER: And histologically are there white cells starting to plug up the microcirculation?

MR LAM: We don’t see an accumulation of neutrophils or microthrombi. So it appears all the events are related to disruption of the endothelial NO pathway. We’re beginning to analyze the microcirculation endothelial cells further using electron microscopy. We additionally are looking at endothelial cell activation markers and markers of apoptosis; however, we haven’t yet finished compiling the data.

DR MAYER: And the last question is, have you tried supplementing the L-arginine concentrations? In old studies that we did many years ago looking at myocardial preservation and the role of endothelial events or endothelial dysfunction, among the most powerful protectors after a period of ischemia was an infusion of L-arginine. And we assumed, although could never prove, that that was because the endothelium was actually making a lot more nitric oxide, and there clearly was endothelial dysfunction in those experimental preparations.

So I just wondered if you or anyone else has tried to actually infuse L-arginine in this post-bypass period in particular to see whether or not it makes any difference.

MR LAM: That is an excellent suggestion. However, in our laboratory’s previous experiments, we haven’t tried to infuse L-arginine after bypass, and I don’t believe that infusion of L-arginine has been previously reported in the literature for fetal bypass.

DR JEFFREY P. JACOBS (St Petersburg, FL): Well, I would just like to congratulate you. To be an undergraduate, stand up in this room, give a presentation like that, and then answer a series of questions from the president of the STS is absolutely truly impressive.

	Acknowledgments

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The authors gratefully acknowledge the technical assistance of Mr Lawrence Spezzano and Dr Keith Stringer, and they thank Dr Anoop Brar for insightful discussions and input. Their research was supported by grants from the American Heart Association, the Children’s Heart Foundation of Chicago, and the Children’s Heart Association of Cincinnati.

	References

Top Abstract Introduction Material and Methods Results Comment Discussion Acknowledgments References

 

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