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Maternal and Fetal Health Research Centre,3 Division of Human Development, University of Manchester, Manchester M13 0JH, United Kingdom
Department Obstetrics and Gyneacology,4 University of Alberta, Edmonton, Alberta, Canada T5H 3V9
Division of Cardiovascular and Endocrine Sciences,5 University of Manchester, Manchester M13 9PT, United Kingdom
ABSTRACT
Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that has been implicated in cardiovascular disease. The objective of the present study was to determine the vasoactive effects and underlying mechanisms of S1P on adult human maternal arteries. The isometric tensions of the omental and myometrial arteries isolated from normal pregnant women at term were assessed in response to incremental doses of S1P in the presence or absence of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). The putative involvement of Rho-associated kinases (ROCKs) in intact arteries and in those permeabilized with
-toxin, to study agonist-dependent calcium-sensitization, was assessed with the inhibitor Y27632. Real-time RT-PCR established the presence of mRNA encoding the S1P receptors (S1P1 to 3), previously known as endothelial differentiation gene receptors (EDG1, 3 and 5), in both artery types. S1P induced a dose-dependent increase in the isometric tension of all the arteries. Y27632 reduced constriction due to S1P in intact arteries and reduced S1P-induced sensitization of contraction to submaximal activating Ca2+ in permeabilized arteries. L-NAME also modulated S1P vasoactive responses in a tissue-specific manner. Two subgroups of omental arteries were identified, one of which utilizes the NO pathway. In myometrial arteries, S1P evoked oscillatory constrictions, whereas pretreatment with L-NAME resulted in only tonic constrictions of unaltered peak magnitude. The prominent vasoactive actions of S1P in the maternal arteries of pregnant women are modulated by inhibitors of ROCKs and NO bioavailability. The subtle tissue-specific functional differences in the modulation of S1P actions by NO have important implications for vascular tone regulation by this bioactive circulatory metabolite during pregnancy.
nitric oxide, Rho-associated kinase, vasoconstriction
Sphingosine-1-Phosphate (S1P) is a bioactive metabolite formed by degradation of sphingomyelin involving the enzyme sphingosine kinase. A major circulating source of S1P is that released from activated platelets: this cell type contains active sphingosine kinase, but no S1P degrading enzyme, S1P lyase. S1P is now recognized as an important regulator of many key cellular processes. In recent years, this molecule has been shown to play roles in the regulation of cell growth, motility, survival, differentiation, proliferation, cytoskeletal organization, and calcium signaling [1]. S1P is known to have intracellular actions in addition to extracellular signaling capabilities. The mechanisms of intracellular signaling are largely unknown, as an intracellular target has not yet been identified, although this is widely recognized as a likely form of action [2]. Extracellularly, S1P is known to act via a family of five G-protein-coupled receptors, known originally as the endothelial differentiation gene (EDG) receptors 1, 3, 5, 6, and 8 (HUGO Gene Nomenclature Committee), but now also known as S1P1–5 (S1P1/EDG1, S1P2/EDG5, S1P3/EDG3, S1P4/EDG6, and S1P5/EDG8). With the exception of EDG1/S1P1, which activates Gi exclusively, the S1P receptors interact with a variety of G-proteins, which contribute to the pleiotropic nature of S1P effects [3–5].
S1P has been shown to have a role in the regulation of vascular tone of different vessel types in many animals [6–16]. The vasoconstrictive effects of S1P, in many cases, have been shown to be dependent upon the activation of EDG 3/S1P3 and EDG5/S1P2 [11, 13–15], while EDG1/S1P1 and EDG3/S1P3 have been shown in larger conduit vessels to be responsible for S1P-induced vasodilation [16]. The tissue distribution of the receptors and the activities of the downstream components of the specific receptor pathways are thought to determine the functional outcomes of the actions of S1P in a given cell or tissue type. The presence of S1P receptors in blood vessels has been determined in animal cerebral and coronary arteries (EDG1, 3 and 5/S1P1 to 3) [11], in which vasoconstriction has been shown to occur via the activation of EDG3 and 5/S1P2 and 3 [11, 13], and in human placental arteries (EDG1, 3, 5, 6, 8/S1P1 to 5) [17]. Vasoconstriction in response to S1P administration is thought to require the Rho-associated kinase (ROCK) pathways, and several studies have used permeabilization of cells within the vessels or the Rho-associated kinase 1 and 2 (ROCK1 and ROCK2) inhibitor Y27632 to demonstrate this mechanism of S1P action [10–13, 17]. Furthermore, it has been reported that Edg5/S1P2 –/– mice show reduced activation of RhoA [18]. A modulatory role for nitric oxide (NO) has also been demonstrated in vessels constricted by S1P [6, 14, 17, 19].
In vivo studies have confirmed the constrictive actions of S1P, its administration in a range of doses caused decreased cerebral (0.3 mg/kg) [13] and coronary (0.01–10 µg) [20] blood flow and reduced myocardial perfusion (38 µg/kg) [21] in various animal models. These experiments lend further evidence to the importance of S1P as a circulating vasoactive lipid. Little is known about the effects of S1P on the human vasculature, although our recent investigation of the effects of S1P on the vascular tone of human arteries of fetal origin showed a dose-dependent increase in vascular tone. This is an important finding and may suggest a role of this metabolite in the pathogenesis of pregnancy complications where inflammation occurs due to its storage and release from platelets.
Pregnancy presents a unique challenge to the human cardiovascular system, whereby greatly increased cardiac output and circulatory volume are needed to provide maternal-placental perfusion sufficient for fetal growth and development. This necessitates functional remodelling of the arteries of the uterus and systemic organs, which are regulated in part by altered responses to circulating vasoactive factors. Although we have ascertained a possible role for S1P in regulating human fetal placental vascular tone, its vasoactive influence on the arteries of the adult maternal circulation is unresolved.
In the present study, we have investigated the functional effects of S1P administration on human arteries isolated from two maternal vascular beds: 1) the myometrium, which gives a measure of the vasoactive effects in the uterus during pregnancy; and 2) the omentum, which allows determination of S1P effects on the systemic vasculature during pregnancy. The contributions of the ROCK1 and ROCK2 pathway, calcium sensitization, and NO to S1P-induced vasoactive actions in the human arteries have been assessed. In addition, the expression of the mRNAs for EDG1, EDG3, and EDG5/S1P1 to 3 have been assayed by Real-Time RT-PCR. Our findings have both physiologic and potentially pathophysiologic importance for the regulation of human vascular tone by circulating metabolites.
Omental and myometrial biopsies were obtained from women with uncomplicated pregnancies, who were undergoing elective Cesarean section at term (37–41 weeks of gestation). The study was approved by the ethical committee of central Manchester Healthcare Trust for implementation at St. Mary's Hospital. Biopsies were obtained following written informed consent, in compliance with the Helsinki Declaration.
Biopsies were placed in modified Krebs solution (pH 7.4) that contained 1.2 M MgSO4, 10 M MOPS (both from Sigma-Aldrich, Poole, UK), 154 mM NaCl, 5.4 mM KCl, 1.6 mM CaCl2, and 5.5 mM glucose (VWR, Lutterworth, UK). Biopsies were then dissected in order to remove the small arteries (150–400 µm in diameter). Omental arteries were identified by comparison to the adjacent vein, which is larger in diameter and has a thinner smooth muscle layer. Myometrial arteries were recognized on the surface of the biopsy and followed into the uterine tissue, where 2–3-mm segments were dissected. Some vessel fragments (10–30 mg) were snap frozen in liquid nitrogen and stored at –80°C until RNA extraction for subsequent Real-Time RT-PCR.
Total RNA Extraction, Ribogreen Quantification, and cDNA Synthesis
Artery samples (10–30 mg) from biopsies of the omentum or myometrium were stored at –80°C. The samples were homogenized and total RNA was extracted using Absolutely RNA (Stratagene, La Jolla, CA). The samples were treated with 5 µl of RNase-free DNase I (Stratagene), in order to prevent contamination with genomic DNA. RNA was quantified using Ribogreen (Molecular Probes, Invitrogen, UK) and the MX4000 thermal cycler (Stratagene). First-strand cDNA synthesis was performed on 100 ng of each RNA sample using random primers (300 ng) (Stratagene). In addition, reverse transcription was performed on human reference RNA samples (Stratagene) (100 ng) in quadruplicate; each of these samples was used subsequently in separate PCR reactions and comparisons of the levels of target gene expression were made between the samples, as a measure of reverse transcriptase efficiency. Human reference RNA (5 µg), which was diluted for PCR to derive the standard curve, was also reverse-transcribed. A sample without reverse transcriptase and a positive (human kidney) control were also added [22, 23]. All samples were stored at –20°C.
The basic procedure used to quantify a gene of interest has been described previously [24]. Briefly, all samples were run in quadruplicate in a final volume of 25 µl, which contained 1 µl cDNA of the sample of interest and Brilliant SYBR Green master mix (Stratagene); 5-carboxy-x-rhodamine (ROX) was also included (30 nM) as a passive reference dye. Previously published primers for human EDG1, 3, and 5/S1P1 to 3 (Table 1) [25] were used to detect and quantify the genes of interest and were included in each reaction at a concentration of 0.5 µM. The amplification conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec, 60°C for 1 min, and 72°C for 30 sec, with a final step of 95°C for 1 min. A dissociation curve was generated beginning at 55°C and increasing by 1°C for 41 cycles of 30 sec each.
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For each transcript, the change in fluorescent signal (
Rn) was plotted against PCR cycle number (Fig. 1A). The cycle threshold (Ct) is calculated as a function of the background fluorescence and is the point at which the signal generated is higher than the background fluorescence. Ct is directly proportional to the amount of starting material. Samples were only used in the subsequent analysis if the efficiency of the sample was determined to be in the range of 93% to 105%. This was calculated using the standard curve (two-fold serial dilutions of human reference RNA) and plotting Ct as a function of log[10] concentration of template, the resulting trend line being a function of efficiency and a slope of –3.32 being equivalent to 100% efficiency (Fig. 1B). The Ct values for each unknown sample are presented as proportions of the calibrator samples.
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Four fresh segments from the same length of artery were mounted in a wire myograph (Danish Myotech, Aarhus, Denmark). Two 40-µM wires were inserted through the lumen of each vessel and attached to the jaws of the bath, with one jaw attached to a micrometer and one attached to a transducer. The vessels were bathed in 6 ml PSS (35 mM NaHCO3 [Fisher Scientific, Loughborough, UK], 2.4 mM MgSO4, 1.18 mM KHPO4 [Sigma-Aldrich], 127.76 mM NaCl, 4.7 mM KCl , 0.03 mM K2EDTA, 5.5 mM glucose, and 1.6 mM CaCl2 [VWR]) in the chambers of a 610M myograph. The Myodaq software version 2.2 (Danish Myotech, Aarhus, Denmark) was used to normalize the arteries at the beginning of each experiment; this determined the vessel stretch needed to generate a tension equivalent to 0.9 of L13.3 kPa, whereby L13.3 kPa is the vessel diameter at which the active effective pressure is 13.3 kPa. Following normalization, the vessels were equilibrated at 37°C for 20 to 30 min. Vessel tone was measured and the raw traces are shown in units of mN/mm, which were subsequently converted into Active effective pressure (kPa) taking into account the diameter of the vessel.
Intact Arterial Contractile Studies
Endothelium integrity, which was defined as a decrease in vessel tone equal to or greater than 70% following the addition of 1 µM bradykinin (Sigma-Aldrich) to a U46619-induced constriction (1 µM) (EMD Biosciences), was evident in all the omental arteries (n = 13) and myometrial arteries tested (n = 6) (data not shown). This was to ensure that the data from our experiments were not skewed by dysfunctional endothelia.
Intact myometrial and omental arteries were validated in experimental arteries by the application of a solution of 120 mM KCl (High K+) and observation of a constriction equal to or greater than 5 kPa. Following PSS washes, all the vessels were bathed in PSS that contained 0.1% BSA (vehicle for S1P). In addition, two of the four vessels were incubated with 100 µM N(G)-nitro-L-arginine methyl ester (L-NAME) (Sigma-Aldrich) for 20 min. Subsequently, all of the vessels were exposed to S1P (Biomol, Exeter, UK) in incremental doses (0.01–40 µM). The effect of the Rho-associated kinase 1 and 2 (ROCK1 and ROCK2) inhibitor Y27632 (EMD Biosciences) at 10 µM was examined in one S1P-incubated vessel and one vessel that was exposed to S1P and L-NAME. The paired time-control vessel in each case was not exposed to Y27632 but was maintained at the maximum S1P concentration.
The protocol used for the permeabilization of human blood vessels mounted on a wire myograph was similar to that described previously [26], with four arterial segments studied in parallel on a single myograph at 25°C. Briefly, as with the intact vessels, constriction with High K+ PSS was performed to validate the vessels. The arteries were then exposed to a mock intracellular relaxing solution (10 mM sodium creatine phosphate, 5.2 mM Na2ATP, 7.3 mM magnesium methanesulphonate, 74 mM potassium methane sulphonate, 1 mM K2EGTA, buffered to pH 7.1 with 30 mM PIPES and KOH) prior to permeabilization with -toxin (EMD Biosciences). The relaxing solution was removed prior to incubation of each vessel with a submaximal concentration of calcium (pCa6.7; the level of free calcium was maintained by the ratio of K2EGTA to Ca2+EGTA) that contained 500 U/ml -toxin and 10 µM A23187 ionophore (EMD Biosciences). The incubation period was deemed to be complete when the resulting submaximal constriction reached a plateau, or in the case of small constrictions, when 30 min had elapsed. Following permeabilization, the vessels were washed in a low-calcium (pCa9) solution. The vessels were then exposed to a high-calcium (pCa 4.5) solution, and the ensuing constriction was monitored. The pCa9 solution reduced the constriction to baseline. A submaximal calcium solution (pCa6.7 with 5 µM GTP) was added after 10 min, producing a small constriction. The agonist (10 µM S1P or 1 µM U46619) was then added to the vessel. Any additional tension development following agonist addition reflected sensitization of the contractile apparatus to the submaximally activating pCa6.7 plus GTP solution. This sensitization in two vessels exposed to S1P was compared to the sensitizing constriction induced in two other vessels run in parallel with U46619, which is a thromboxane mimetic that is known to induce prominent Ca2+ sensitization [26]. The magnitudes of the constrictions induced by S1P (10 µM) or U46619 (1 µM) over and above those induced by pCa6.7 plus GTP were measured as proportions of the constriction produced upon previous exposure to pCa 4.5 alone in the same vessel. The ROCK1 and ROCK 2 inhibitor Y27632 (10 µM) was applied to one S1P-constricted and one U46619-constricted vessel segment. The two other segments were retained in S1P or U46619 in the absence of Y27632, to serve as time controls.
The expression of S1P receptor mRNA was analyzed using the Kruskal-Wallis test with Dunn multiple comparison post-hoc tests. The data from dose-response curves were log-transformed to enable use of the parametric two-way ANOVA test with Bonferroni multiple comparison post-hoc analysis. The vessel diameters before and after treatment with Y27632 were compared with the Mann-Whitney test. P < 0.05 was taken as indicating statistical significance.
Expression of EDG1, EDG3, and 5/S1P1 to 3 in Human Omental and Myometrial Arteries
Using Real-Time RT-PCR, both the omental (n = 11) and myometrial arteries (n = 11) were shown to express the S1P receptor mRNAs for EDG1, 3, and 5/S1P1 to 3. The medians were compared using a Kruskal-Wallis test. EDG1/S1P1 was found to be the predominant receptor expressed in both the omental and myometrial arteries (Fig. 2). The Dunn multiple comparison test revealed that the mRNA for EDG1/S1P1 in omental arteries was significantly more abundant than the mRNAs for EDG5/S1P2 (P < 0.001) and EDG3/S1P3 (P < 0.05). In addition, the expression of EDG1/S1P1 in myometrial arteries was found to be significantly higher than those of EDG3 and 5/S1P2 and 3 (P < 0.001). All three receptor subtypes were expressed at higher levels in the omental arteries than in the myometrial arteries.
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S1P-Induced Constriction of Intact Omental Arteries Ex Vivo
The responses of omental arteries to increasing concentrations of S1P involved dose-dependent constriction (Fig. 3A) to a maximum of 6.6 ± 1.96 kPa (n = 9). Pretreatment with L-NAME had no significant overall effect on these responses. However, two distinct functional responses to S1P stimulation were observed within the omental vasculature. One group produced a maximum constriction to S1P (40 µM) that was comparable to that of the preceding High K+-induced constriction (group 1; S1P constriction >20% of the High K+-induced constriction; n = 5), while the other group produced a maximum S1P-induced constriction of a far smaller magnitude compared to the constriction induced by High K+ (group 2; S1P constriction 
20% of the High K+-induced constriction; n = 4) (Fig. 3B). The vessel segments categorized in group 1, which produced a constriction of similar magnitude to that of High K+-induced response to the maximum S1P dose of 9.3 ± 2.7 kPa, exhibited no differences when fragments from the same length of vessel were exposed to 100 µM L-NAME (7.4 ± 1.6 kPa at the maximum dose of S1P) in parallel myograph baths (Fig. 4, A, C, and E). However, the weak S1P-induced constrictions produced by the vessels in group 2 (20% of the High K+-induced response) were significantly (P 0.05) enhanced by preincubation with L-NAME (increased from a maximum of 1.25 ± 0.24 kPa to a maximum of 4 ± 1.1 kPa in the presence of L-NAME) (Fig. 4, B, D, and F). Irregular oscillations were observed in some vessels from both the L-NAME-pretreated and vehicle-treated control groups (Fig. 4, A–D).
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The S1P-induced constrictions from both functional groups, whether pre-treated with L-NAME or not, were reversed by the addition of the ROCK1 and ROCK2 inhibitor Y27632 (10 µM) to 7.3% (0.46 ± 0.2 kPa) and 11.9% (0.73 ± 0.5 kPa) of the maximum constriction, respectively (Fig. 3C and Fig. 4, A–D).
S1P-Induced Constriction of Intact Myometrial Arteries Ex Vivo
The responses of myometrial arteries could not be separated into two groups, with the constriction in response to 40 µM S1P being greater than 20% of the High K+-induced constriction in every case (Fig. 5A). The myometrial arteries produced a dose-dependent constriction to increasing concentrations of S1P, to a maximum amplitude of 8.6 ± 1.6 kPa (79.5% of High K+-induced constriction) (Fig. 5, B and D). Myometrial arteries that received 100 µM L-NAME treatment prior to S1P application showed no significant changes in constriction at any dose of S1P (0.01–40 µM) to a maximum of 9.3 ± 3.1 kPa (69% of the High K+-induced constriction) (Fig. 5, C and D).
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It is pertinent to note that in every case, the S1P-induced response of myometrial arteries was associated with regular large oscillations in tone, which were absent in vessels that were pretreated with L-NAME (Fig. 5, B and C).
The ROCK1 and ROCK2 inhibitor Y27632 (10 µM) reduced the S1P-induced constriction in myometrial arteries, whether pre-treated with L-NAME or not, to 13.9% (1.3 ± 0.4 kPa) and 40.65% (3.8 ± 1.9 kPa) of the maximum constriction, respectively (Fig. 5, B, C, and E).
S1P-Induced Responses in Permeabilized Arteries
Given the potential role of ROCKs in calcium sensitization of contraction of smooth muscles and the effect of the ROCK1 and ROCK2 inhibitor on the S1P-induced constriction of intact human arteries, it is possible that S1P effects an increase in the tone of these vessels through a similar mechanism. To investigate this notion, arteries were permeabilized with -toxin, to allow rapid equilibration of the extracellular and intracellular calcium concentrations. Figure 6 shows traces of agonist-induced constrictions in permeabilized human omental arteries (Fig. 6, A–D). The S1P-induced (10 µM) constriction (1.4 ± 0.2-fold change compared to pCa 4.5) was of a similar magnitude to that produced by 1 µM U46619 (1.3 ± 0.1-fold change compared to pCa 4.5) (Fig. 6, A–E), which demonstrates sensitization of the contractile myofilaments by both agonists to the pCa 6.7 solution. Y27632 significantly reduced these constrictions to 11.9% (0.3 ± 0.2-fold change) and 7.3% (0.2 ± 0.06-fold change), respectively (Fig. 6, B, D, and E). The control traces (Fig. 6, A and C) show that this is not a time-dependent effect.
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These data are the first to show the potent vasoactive effects of S1P in human maternal arteries during pregnancy. The study suggests the involvement of ROCKs and NOS in downstream signaling of S1P-induced changes in vascular tone in both omental and myometrial arteries. This is in agreement with previous studies in a variety of animal vessel types [6, 9–13, 27] and human placental arteries [17], which have also implicated these molecules in the regulation of S1P-induced vasoactive actions. Interestingly, the mechanism of action differs between the vascular beds of the omentum and myometrium, with reduced involvement of NOS in the magnitude of S1P-induced constriction in myometrial vessels compared to omental arteries (group 2). In addition, our data illustrate the involvement of NO in the regulation of S1P-induced oscillations in myometrial arteries. In contrast to omental arteries, large regular oscillations were produced in all myometrial arteries exposed to S1P, and pretreatment with L-NAME abolished these oscillations, demonstrating the importance of NO in this process [28].
There was also evidence of differential mechanisms of S1P action within the omental vasculature. Approximately half of the omental arteries produced constriction to S1P of a similar magnitude to that of the High K+ control, and this constriction was not enhanced by L-NAME preincubation. The remaining arteries generated a very small response to S1P administration, which was greatly enhanced by preincubation with L-NAME. Since no similar pattern of function was seen in similar arteries treated with other constrictive agents that use the ROCK1 and ROCK2 pathway, e.g., arginine vasopressin [29], this response is not due to variability of vascular function during pregnancy. Our findings indicate two functional subgroups, one that requires NO modulation for its response to S1P and the other that is independent of NO modulation. The data point to a complicated scenario, possibly entailing a combination of signaling pathways that involve direct and indirect interactions with S1P and its receptors. Receptor distribution or location may explain the variety of mechanisms observed.
It has been suggested that the level of constriction to S1P may depend on the balance of receptor subtypes between the smooth muscle cells and the endothelial cells [30]. EDG3 and 5/S1P2 and 3 are involved in the constriction of many animal vessels [11, 13], by activation of the ROCK pathways and calcium sensitization. Others have shown that EDG1 and 3/S1P1 and 3 are involved in the dilation of conduit vessels, such as the aorta [16], which involves NO modulation. Therefore, the conflicting actions of S1P receptors located on different cell types may produce the net response to S1P in a given vessel. We speculate that S1P receptor expression and distribution may influence the effect of S1P stimulation in maternal arteries. Therefore, the different responses to S1P observed between and within vascular beds may be explained by this hypothesis. However, whole vessel mRNA S1P receptor expression analysis has failed to resolve this question. These data do not distinguish between vascular cell types (smooth muscle cells and endothelial cells) and it is not certain that the transcripts are translated into functional proteins. Therefore, further investigations to determine the localizations of these three S1P receptor subtypes are required. Cell-specific expression analysis would be useful in conjunction with protein localization. Unfortunately, obtaining pure cell populations for RNA analysis from these tiny human vessel segments is technically difficult and the commercially available antibodies to EDG1, 3, and 5/S1P1 to 3 have been unreliable in our hands. A potential way to answer this question is to denude the arteries before purification of the RNA and real-time RT-PCR. This would remove endothelium-derived RNAs, and when compared with intact arteries, would indicate whether the differences between these two groups are due to differential expression of genes between the endothelial and smooth muscle cells. A more precise method of isolating these cell types from arteries would be mechanical dissection of tissue sections to remove pure populations of cells, a technique that has been used by other groups [31].
The actions of S1P may also be of pathophysiologic importance. S1P is thought to be elevated during inflammation due to platelet activation and aggregation, which would produce higher local levels of S1P. S1P is known to be induced by sphingosine kinase (SPHK). SPHK, in turn, has been shown to be activated by growth factors and cytokines, such as platelet derived growth factor (PDGF) [32] and tumor necrosis factor (TNF) [19, 33]. Therefore, under pathologic conditions in which inflammation is associated with hypertension, it may be particularly pertinent to consider the contribution of S1P-induced vasoactive actions. In particular, the regulation of vascular tone in both the fetal-placental and maternal circulations is essential for normal pregnancy outcome. In the future, S1P studies will be carried out in arteries from women with pregnancy complications, such as hypertension, pre-eclampsia, and intrauterine growth restriction (IUGR), which are associated with impairments of the maternal and placental circulation [34, 35].
In summary, we have shown potent vasoactive effects for S1P in human myometrial and omental arteries from pregnant women at term. Pathways that involve NO and ROCKs are implicated in the actions of S1P, although these pathways are differentially regulated depending on the vascular bed.
ACKNOWLEDGMENTS
We thank Dr. Michèle Sweeney for advice on experimental matters, and the doctors, midwives and patients at St. Mary’s hospital for their cooperation during this study.
FOOTNOTES
1Supported by Ardana Bioscience UK and the Canadian Institutes of Health Research (CIHR). 
Correspondence: 2Nicola Hudson, Maternal and Fetal Health Research Centre, St Mary's Hospital, Hathersage Road, University of Manchester, Manchester M13 0JH, United Kingdom. FAX: 44 0161 276 6133; e-mail: nicola.hudson{at}manchester.ac.uk
Received: 7 February 2007.
First decision: 23 February 2007.
Accepted: 3 April 2007.
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