INVITED REVIEW
Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function

¹ University of Wisconsin-Madison, Department of Obstetrics and Gynecology, Perinatal Research Laboratories and the ² Department of Pediatrics and ⁴ Animal Sciences, Madison, Wisconsin 53715; and ³ Center for Perinatal Biology, Department of Pharmacology and Physiology, Loma Linda University School of Medicine, Loma Linda, California 92350

	ABSTRACT

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The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or preeclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am J Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PGI₂) may be as much a consequence of cellular reprogramming at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PGI₂ biosynthetic pathway (cPLA₂, COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant.

nitric oxide; prostacyclin; calcium; kinases; programming

	INTRODUCTION

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OUR UNDERSTANDING OF THE MECHANISMS underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus has increased dramatically in the last decade. The importance of these changes is clear from a number of studies.

	IN VIVO PREGNANCY-INDUCED CHANGES IN CARDIOVASCULAR FUNCTION AND BLOOD FLOW REDISTRIBUTION TO THE UTERUS

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Maternal cardiovascular adaptations observed during normal pregnancy include decreases in uterine and systemic vascular resistance with dramatic increases in uterine blood flow (UBF), cardiac output via changes in heart rate and stroke volume, and rises in blood volume (reviewed in Refs. 61 and 74). Development of systemic and uterine vascular refractoriness to the vasoconstrictor effects of several agents, including ANG II and norepinephrine (64, 67, 68, 88), is also observed. The relative decrease in uterine vascular resistance greatly exceeds the relative fall in systemic vascular resistance (61, 62, 74, 99, 101), and the uterine vascular bed is even less reactive to the vasoconstrictor effects of ANG II than the systemic vasculature (23, 63, 70). In contrast, the uterine vascular bed is substantially more responsive to -agonists such as norepinephrine and phenylephrine than the systemic vasculature overall (68). This results in a 20- to 50-fold rise in ovine UBF, so delivering oxygen and nutrient substrates via the placenta to meet the metabolic demand of fetal growth and development.

The exact mechanisms of the rise in uteroplacental perfusion and accompanying decrease in vascular reactivity remain unclear. Although growth of new vessels as well as remodeling of existing vessels during early pregnancy contributes to the increased UBF, the fact that the period of greatest increase in UBF occurs after the completion of new vessel growth indicates that the maintenance of vasodilation in existing or newly developed vessels is crucial. Herein we focus on the endothelium-derived vasodilators that are profoundly elevated during pregnancy and most studied to date, i.e., NO and PGI₂, rather than changes in vasoconstrictors that are less well understood. An in vivo physiological role for endothelium-derived NO and PGI₂ as a direct modulator of vascular smooth muscle (VSM) tone and normal cardiovascular adaptation during pregnancy is supported by the following findings.

In Vivo

Plasma levels of nitrates/nitrites (NOx) and 6-keto-PGF₁, the stable metabolites of nitric oxide (NO) and PGI₂, are increased during pregnancy, suggesting that endogenous vascular NO and PGI₂ production is increased in gravid animals (13, 73, 130, 135). PGI₂ production is elevated during normal pregnancy in women and reduced in preeclampsia, suggesting important clinical significance to endothelial activation in pregnancy and dysfunction in diseased states (26, 33). NOx have also been reported to be elevated in human pregnancy (94, 107), although this observation has subsequently been questioned in women where dietary intake of NOx is controlled (14). Nonetheless, inhibition of NO with N^G-nitro-L-arginine methyl ester (L-NAME) or PGI₂ with indomethacin in some studies decreases UBF and enhances systemic and uterine vasoconstrictor responses to several vasoconstrictors, including ANG II (24, 66, 70, 78, 100, 116). Reversal of the vascular refractoriness to vasopressor agents in pregnant rats (1, 84) is associated with decreased fetal birth weights (129). Positive uterine venous-arterial concentration differences of cGMP and 38-fold increases in uterine cGMP secretion have been reported during late ovine (100) and human (14) gestation, indicating that the uteroplacental unit (including its vasculature) may be responsible for a substantial portion of this rise in circulating cGMP in pregnancy (via elevations in uterine endothelium-derived NO). Positive venous arterial concentrations of NOx in ovine (72, 130) and human (14) pregnancy have proved undetectable, but the long half-life of this metabolite may have masked the overall rise in NO. Nonetheless, progressive increases in NO production, measured as NO₂/NO₃ (13, 18, 107, 130, 135), are associated with rises in cGMP as well as NO bound hemoglobin (13), which in turn are decreased by L-NAME (18).

Ex Vivo

Basal and agonist-stimulated NO and PGI₂ production ex vivo is increased in UA from pregnant animals and is produced at the level of the UA endothelium (63, 65, 71, 108, 126, 128). Increased agonist-sensitive NO and PGI₂ production from vascular endothelium is seen to cause corresponding VSM cGMP and cAMP production, respectively (24, 66, 78, 109), consistent with the elevated circulating and urinary cGMP and cAMP observed during normal pregnancy (13, 54, 71, 100).

	EX VIVO STUDIES OF PREGNANCY-INDUCED CHANGES IN UA FUNCTION

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The above findings suggest that maternal cardiovascular alterations in pregnancy occur through changes in the paracrine interaction between the cells comprising the vessel wall, i.e., endothelial and VSM cells. The use of sodium nitroprusside treatment to mimic NO production also relaxes precontracted UA obtained from nonpregnant and pregnant women (89), guinea pigs (118), rats (92), and sheep (63, 109, 126). In vitro relaxation responses to sodium nitroprusside (SNP) were, however, similar in UA from nonpregnant and pregnant ewes (89, 92, 109, 126), suggesting that differences in uterine vascular reactivity during pregnancy may not be mediated by increased sensitivity of uterine VSM to NO, even though greater guanylate cyclase immunostaining (Western and immunohistochemical) has been observed in pregnant vs. nonpregnant sheep (44).

Bell (5) was the first to demonstrate that the in vitro perfused precontracted uterine vascular bed from pregnant, but not nonpregnant, guinea pigs vasodilates in response to ACh. ACh relaxation was greater in UA from pregnant vs. nonpregnant guinea pigs, rats, and women (91, 109, 118). In addition, endothelium-dependent relaxation induced by ACh was enhanced significantly in UA rings from pregnant guinea pigs compared with those from nonpregnant animals (119). Although ACh is the prototype endothelium-dependent vasodilator and has often been assumed to act by stimulating NO release, other studies suggest that ACh-induced vascular relaxation may not be mediated solely by NO synthesis/release (3, 80). Further studies using isolated vessels have also shown that contractile responses to norepinephrine in pregnant compared with nonpregnant UA were increased by removal of the endothelium or by blocking of NO synthesis with N^G-monomethyl-L-arginine (L-NMMA; 118, 121). Both NO and PGs (most likely PGI₂) are implicated in the pregnancy-associated uterine vascular refractoriness of UA, because both L-NMMA, which inhibits NO synthase (NOS), and indomethacin, which inhibits COX, shifted the norepinephrine dose-response curves of pregnant guinea pigs to the left (i.e., more like those of the nonpregnant). Whereas these early studies have all evaluated the effect of pregnancy on UA NO release by means of endothelium removal and NOS inhibitors on either vasoconstrictor responses or vasorelaxation responses of precontracted vessels (for review see Ref. 109), more recent studies of perfused ovine UA are noteworthy in that they are the first to demonstrate directly both basal and agonist (ATP and the calcium ionophore A23187)-induced endothelial NOx release were significantly increased in pregnant UA (125, 126, 128).

	MECHANISMS UNDERLYING CHANGES IN UA ENDOTHELIAL FUNCTION

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Changes in Expression of Genes in Vasodilator Pathway

Changes observed in eNOS, cPLA₂, and COX-1 expression. Activity assays in UA homogenates (with vs. without endothelium) were among the first data to show a Ca²⁺-sensitive, endothelium located NOS is elevated in pregnant vs. nonpregnant guinea pigs (118), sheep (58, 65), and, more recently, humans (90). In sheep, this increase was specific to the UA because no difference was noted in NOS activity among omental arteries due to pregnancy (65). In addition, the expression of eNOS protein and mRNA is increased by pregnancy in ovine (8, 46, 71, 125) and human (90) UA endothelium. More recently, studies in sheep have demonstrated that the pregnancy-associated increase in the endothelium-dependent relaxation of the UA is mediated predominantly by the upregulation of NO release resulting in a decrease in smooth muscle intracellular free Ca²⁺ ([Ca²⁺]_i). At the level of VSM, signal transduction pathways downstream of NO are largely unaltered, confirming the pregnancy-specific change is at the level of endothelial NO production (126). Normal pregnancy is also directly associated with a parallel increase in UA endothelial cPLA₂ and COX-1 protein (8, 20, 36, 45) and, to some extent, a corresponding COX-1 mRNA (8, 36, 45). Together these protein expression changes are substantial in the uterine vascular bed and are not just the result of general increases in all cellular proteins because changes in eNOS and COX-1 (8, 36, 45, 71, 73) greatly exceed lesser but otherwise significant changes in cPLA₂ and PGI₂ synthase (PGIS) (8, 20, 72). Thus there is a coordinated increase in the capacity of UA to resist vasoconstriction by agonists such as ANG II and ATP via increases in endothelial vasodilator production.

We recently reported in two physiological states of high UBF and elevated estrogen, i.e., pregnancy and the follicular phase of the ovarian cycle, that the UA endothelial eNOS and COX-1 protein and mRNA levels are dramatically elevated compared with luteal phase controls (36, 45, 71, 73). A physiological role for estrogen in this response derives from recent unpublished data showing that infusion of the estrogen receptor antagonist ICI-182,780 decreases UBF in pregnant sheep (RR Magness and TM Phernetton, unpublished observations). The levels of eNOS and, to a lesser extent, COX-1 protein in the UA endothelium were also elevated in ovariectomized sheep treated with exogenous estrogen with or without progesterone (102, 103, 114, 115). A comparison of the effects of pregnancy and steroid treatment (10 days) in vivo (summarized in Table 1) indicates differential mechanisms regulate expression of the enzymes involved in PGI₂ vs. NO biosynthesis in UA endothelium in particular (20, 36, 45, 72, 73, 102, 103). Pregnancy substantially elevates the expression of eNOS and COX-1 and although this is mimicked to a great extent for eNOS by the combination treatment with progesterone plus estrogen, this is not so apparent for COX-1. Because pregnancy is a state of elevated progesterone and estrogen, these data would suggest that these treatments partially recapitulate the effects of pregnancy but also suggest that endocrine and/or molecular mechanisms regulating these two proteins, as well as cPLA₂, differ. This is further supported by the finding that estrogen treatments under these in vivo conditions increase the levels of all the enzymes studied in UA endothelium, whereas progesterone only minimally increases the level of eNOS. Further studies will clearly be necessary to understand the role of additional factors such as shear stress or placental hormones, but differential regulation of the PGI₂ vs. NO pathway automatically implies some ability for one pathway to compensate for a loss of the other under endocrine and/or biochemical imbalance.

View this table: [in this window] [in a new window]   Table 1.   Effects of pregnancy and prolonged (10 days) progesterone and estrogen treatment on the regulation of enzymes that control the production of the vasodilators NO and PGI2 in UA endothelium

At the level of VSM, UA levels of eNOS, cPLA₂, and COX-1, but not PGIS were quite low, and, in some experiments, especially after ovariectomy, not even detectable by Western analysis. In contrast, PGIS levels are quite robust and were marginally increased by pregnancy (72) and only slightly (<1-fold above control) by progesterone in the absence or presence of estrogen (103). These data again suggest that primary changes in the endothelium rather than the VSM explain altered function in pregnancy and that ovarian steroid hormone treatment may be responsible for the changes in vasodilator production in pregnancy. However, our data do not address the overall role of these steroid hormones in conjunction with NO and PGI₂ in the alterations in VSM reactivity, VSM signaling, or VSM remodeling during these physiological states, nor do they address the possible role of shear stress on the endothelium.

Consistent with the findings in Table 1, a cause and effect relationship in vivo of ovarian steroids on eNOS expression and production of NO has been inferred. Uterine vasodilatory response to estradiol-2 (E2) involves the augmentation of NOS enzyme activity as well as the de novo protein synthesis, because, respectively, L-NAME and cycloheximide decrease E2-induced increases in UBF (53, 100, 105, 116, 117). In these studies, L-NAME inhibited 60-70% of the E2-mediated increases in UBF (100, 116). Estrogen treatment also elevated uterine cGMP production, and L-NAME treatments reduced E2-mediated increases in uterine cGMP production (100), the second messenger that mediates the physiological actions of NO in VSM (65, 100). Functional studies showed that 3 days of E2 infusion into ovariectomized sheep increases uterine, but not renal, artery NOS specific activity and NO-mediated endothelium-dependent relaxation (117). These in vivo observations underscore the physiological importance of NOS activation in this UBF response to estrogen and suggest indirectly that E2 acutely (120 min) or chronically (3 days) increases a protein that regulates NOS specific activity or, as we have shown, E2 elevates the de novo expression of NOS protein itself (115); these studies were recently confirmed in the sheep and human (90, 105). The involvement of the PGI₂ pathway is less clear because the mixed COX-1/2 inhibitor indomethacin did not inhibit the E2-mediated increases in UBF (100). However, it is also still unclear if the NOS pathway can acutely compensate for the loss of vasodilator when indomethacin is present.

Further discoveries from the effects of hypoxia on UA function. Chronic uteroplacental hypoxia during the course of pregnancy is one of the most common insults to the maternal cardiovascular system and fetal development and is thought to be associated with increased risk of preeclampsia and fetal IUGR (85, 86, 131). Whereas the regulation of UBF is important, both for growth and survival of the fetus and for maternal cardiovascular well being, the adaptive mechanisms of the uterine vasculature to chronic hypoxia are likely to be species dependent. In pregnant women residing at high altitude (3,100 m) throughout pregnancy, UBF at 36 wk was decreased compared with those at low altitude (1,600 m), due primarily to a decreased vessel diameter resulting from a structural remodeling of the UA (131). In contrast, the blood flow velocity was, in fact, higher in the high-altitude women, which in part helped to compensate for the reduced diameter and may have resulted from vasodilation (131). In the guinea pig, chronic high-altitude hypoxia did not diminish the pregnancy-associated reduction in contractile sensitivity to phenylephrine but enhanced basal nitric oxide activity in the nonpregnant UA and the pregnant mesenteric artery (123). In sheep, chronic high-altitude hypoxia significantly suppressed vasoconstrictor-signaling pathways and decreased UA contractility in pregnant animals (39, 40, 41, 132, 133). The effects of chronic hypoxia on endothelial NO synthesis and release have been investigated both in cultured cells and in vivo, but the results are controversial (4, 57, 59, 79, 96, 111). In rat pulmonary vasculature, chronic hypoxia increased endothelial NO release and up-regulated endothelial (eNOS) and inducible (iNOS) gene and protein expression (42, 57, 98). In contrast, in rat aorta, it has been shown that chronic hypoxia results in a decrease in eNOS protein and mRNA and impaired endothelium-dependent relaxation (111). In a recent study, White et al. (122) demonstrated that ACh-mediated relaxation of the UA of near-term pregnant guinea pig was the same in those animals kept in a hypobaric chamber at a simulated high altitude (3,962 m) throughout gestation as in those kept at low altitude (1,600 m). Nonetheless, the effect of NOS inhibitor N^G-nitro-L-arginine (L-NNA) on the relaxation response to ACh was decreased in the UA of simulated high-altitude pregnant guinea pig compared with the low-altitude controls. On the basis of these findings, the authors suggested that stimulatory effect of pregnancy on NO in the guinea pig was diminished at high compared with low altitude. However, the effect of high-altitude chronic hypoxia on UA endothelial NO synthesis/release and eNOS gene expression was not examined. We demonstrated that long-term (~110 days) moderate high-altitude (3,820 m) exposure increased plasma nitrate levels in the near-term pregnant but not in nonpregnant sheep (135). More recently, we demonstrated that chronic high-altitude hypoxia during the course of pregnancy in sheep increased endothelium-dependent relaxation, basal and agonist-induced NO release and eNOS protein and mRNA in UA endothelium (125). It is unlikely that the increased endothelium-dependent relaxation was due to changes at downstream signals of soluble guanylate cyclase or cGMP-dependent protein kinase in the VSM, because chronic hypoxia had no effect on SNP- or 8-Br-cGMP-mediated relaxation in the UA. The close correlation of the increase in both basal NO release and eNOS protein levels suggest that increased basal NO release is predominantly due to the increased eNOS protein in hypoxic UA. On the other hand, the degree of increase of NO release induced by the calcium ionophore A23187 was far higher than that of eNOS protein expression in hypoxic UA, suggesting a hypoxia-mediated increase in sensitivity of eNOS activation must have occurred at the post-receptor level over and above the effect of enhanced protein expression. The finding that chronic hypoxia increased eNOS mRNA levels but did not change apparent translational efficiency of eNOS mRNA suggests that increased eNOS protein expression in the pregnant UA endothelium may not be regulated at the translational level. In addition, it is unlikely that increased steady-state eNOS protein levels found in hypoxic, compared with control, pregnant UA endothelium were due to increased protein stability because eNOS protein vs. message was constant among different groups. It is not clear from the study to what extent increased steady-state mRNA levels resulted from increased transcription or enhanced message stability.

Changes in Specific Endocrine Systems

Changes in cell capacity vs. cell signaling in regulation of vasodilator production. It is tempting to assume that the pregnancy-induced changes in NO and PGI₂ production can be fully explained by changes in expression of the corresponding biosynthetic enzymes (above), but the situation is in fact more complex. Changes in the expression of any enzyme undertaking the rate-limiting step in a pathway will clearly have the biggest effect on the capacity of the cell to produce vasodilators, but that alone does not mean the pathway's activity will be increased. To understand this it is important to also recognize that both NO production and PGI₂ production require hormone sensitive steps to be activated. Therefore we must not only consider what step is limiting the cell's capacity to make these vasodilators but also consider if the cell has the necessary means to activate these pathways to capacity. For NO production, the rate-limiting and hormone-sensitive step is eNOS itself, whereas in the case of PGI₂ production COX-1 is generally thought to be rate limiting, but it is cPLA₂ that is hormone sensitive. Thus in the basal state, cPLA₂ does not produce substrate for COX-1 but once cPLA₂ is activated by an increase in [Ca²⁺]_i and/or protein kinase activity (below), COX-1 is rate limiting. In the face of incomplete activation, a likely situation in vivo, it is also conceivable that cPLA₂ may remain the rate-limiting step. Once all this is considered then it becomes clear that changes in the expression of eNOS and COX-1, as well as cPLA₂, may well alter the capacity of the cell to make NO and PGI₂, respectively. If, however, the existing cell-signaling apparatus is not sufficient to activate the greater quantity of enzyme, then additional modification of endocrine-signaling mechanisms controlling eNOS and/or cPLA₂ activation may also be necessary. The first level at which this can occur is increased receptor expression. Such changes in receptor expression could achieve greater signaling as long as the receptor number increase does not exceed the capacity of intermediate proteins such as heterotrimeric G proteins or downstream signaling events. In turn, changes in numbers of a specific receptor would be indicated by an increased vasodilator response to a single agonist. The second approach is to make changes at the postreceptor level to alter availability of existing postreceptor G proteins or downstream signaling enzymes/molecules. This would allow the cell to introduce new pathways or alter the balance of opposing systems to achieve even higher levels of activation of the rate-limiting enzymes eNOS or cPLA₂ that were not possible before. The difference in outcome of this compared with changes in receptor levels would be increased vasodilator production in response to groups of agonists acting through these common signaling pathways.

Changes in receptor expression. We have reported that pregnancy-induced increases in endothelial production of NO and PGI₂ in response to ANG II are indeed associated with increased endothelial ANG II type 1 receptor (AT₁-R) expression at the level of both cell protein and mRNA in UA endothelium (8, 9). The increase in AT₁-R protein also greatly exceeds that observed in omental (systemic) artery endothelium (9). We also showed that the specific increase in AT₁-R only in UA endothelial cells (UAEC) was not the result of alternate splicing or promoter usage to generate a unique transcript and so must have been an "endocrine" phenomenon (7). Combined with the associated parallel increase in UA endothelial eNOS, cPLA₂ and COX-1 protein (8, 20, 45, 71), and corresponding mRNA (8, 45, 71), there is a coordinated increase in the capacity of UA to resist vasoconstriction in response to ANG II via increases in endothelial vasodilator production. However, changes in AT₁-R would not explain altered responsiveness to agonists such as ATP (8, 19, 128), and for this reason we have more recently focused our efforts on possible changes in cell signaling.

Studies demonstrating remapping of cell signaling within UA endothelium. Evidence for the ability to remap cell signaling within UA endothelium is implied by several in vivo studies. Studies of expression levels of eNOS protein in UA endothelium show that during the ovarian cycle, the follicular phase is associated with a marginal increase in eNOS expression and systemic NO, whereas pregnancy is associated with a far greater increase in both eNOS protein and systemic NO (73). Further analysis of the UA eNOS levels and systemic NOx levels in pregnancies with singleton, twins, and triplets, however, shows that eNOS does not undergo any substantial further increase with number of offspring, whereas NOx clearly does (73). Also, in ovariectomized ewes in response to exogenous steroid, changes in eNOS expression in response to E2 and P4 alone or together are not uniformly paralleled by changes in systemic NO (102). Whereas these studies suggest dissociation between eNOS protein and function in UA endothelium, the use of systemic NO measurements assumes that uterine NO production is the major determinant of systemic NOx, which may not always be the case. Other studies by Xiao et al. (125) are more clear-cut, because both eNOS level and NOx were measured pairwise ex vivo. Whereas pregnancy increased eNOS protein levels in the UA endothelium and hypoxia increased the expression further still, increases in NOx were still greater than associated increases in eNOS protein. Such observations would be consistent with enhanced cell signaling and, because the agonist used to stimulate NOx production was A23187, an agent that acts independent of receptors, then the change would have to be at the postreceptor level (125).

The clearest evidence for the ability to remap cell signaling in UA endothelium comes from in vitro studies of UAEC in primary cell culture. In maintaining cells from nonpregnant or pregnant UA endothelium to the fourth passage (NP-UAEC and P-UAEC, respectively), the different expression levels of eNOS are greatly reduced (8) and yet the ability of the cells to produce NOx in response to a number of agonists is still clearly different, with P-UAEC production being greater than NP-UAEC (8, 19).

Possible Mechanistic Models for Remapping of Cell Signaling

To identify the level of changes in cell signaling it is first necessary to understand current concepts of the regulation of cPLA₂ and eNOS before reviewing the possible changes that occur in UA endothelium between the nonpregnant and pregnant state. This area has undergone dramatic revision in recent years, and it should be noted that the situation concerning control of cPLA₂ is much better understood than that for eNOS and therefore gives us indirect confirmation of the signal pathways activated in endothelial cells.

Current concepts of regulation of cPLA₂ and eNOS. In initial characterizations of both cPLA₂ and eNOS it was quickly realized that both enzymes require Ca²⁺ for activity, but an enzyme's requirement for Ca²⁺ does not always mean it is physiologically regulated by changes in cytosolic [Ca²⁺]_i. Intracellular [Ca²⁺] typically ranges from 50 to 1,000 nM. If the enzyme's requirement for Ca²⁺ is below or above that range, it will either be permanently activated or never activated by [Ca²⁺]_i elevation, respectively. However, in many cases, with cPLA₂ as an excellent example, posttranslational alterations in protein structure through phosphorylation can result in a dramatic increase in Ca²⁺ sensitivity and so a parallel shift of the [Ca²⁺]_i dose response to the left, which in turn results in enhanced activation. Thus phosphorylation alone can result in a marked increase in activity without additional increases in [Ca²⁺]_i. Studies performed in bovine aorta endothelial cells have shown clearly that this occurs and that cPLA₂ is indeed a substrate for MAPK (104). In addition, site-directed mutagenesis studies have suggested that phosphorylation of amino acid Ser 505 is critical because its elimination negates this response in Chinese hamster ovary cells (60). Thus we can clearly see that cPLA₂ can provide a point of convergence for control of PGI₂ production through agonists that act through mobilization of Ca²⁺ (ATP) with those that activate ERK-1/2 [ANG II and/or basic fibroblast growth factor (bFGF)] in UAEC. In reality, the activation of cPLA₂ by Ca²⁺ and MAPK is more complex, with Ca²⁺ actually triggering membrane binding and so translocation of the enzyme from the cytosol at the basal state to the membrane fraction (more specifically the nuclear envelope and endoplasmic reticulum 106), whereas phosphorylation of Ser 505 is necessary to achieve hydrolysis of the bound lipid in the presence of physiological levels of cytosolic Ca²⁺. An interesting additional point is that the cellular location of cPLA₂, once membrane bound, also correlates with the distribution of COX-1 in Chinese hamster ovary cells, suggesting that cPLA₂ translocation brings it into close proximity with the next enzyme involved in PGI₂ biosynthesis from arachidonate (106).

The situation with eNOS is more complicated than for cPLA₂ and less well understood. It is clear that eNOS can be activated by Ca²⁺ via calmodulin and that subsequent complete/total removal of Ca²⁺ from the media abolishes such activation of eNOS in vitro, but it is also becoming increasingly apparent that either inhibition of tyrosine phosphatases (27), or shear stress (15) can also increase eNOS phosphorylation and activity independently of an increase in [Ca²⁺]_i in endothelial cells (15, 27). The initial model for eNOS activation by Ca²⁺ was as follows. At rest, eNOS is bound to caveolin-1 at the caveolae (plasma membrane invaginations rich in signaling proteins and substrates) and is inactive. Stable association probably also relies on the palmitoylation and myristilation of eNOS as well as binding to caveolin-1. Two events, however, seem able to out-compete or destabilize the binding of eNOS to the caveolin and phospholipid bilayer. The first is competitive binding of Ca²⁺/CaM formed on cytosolic increases in [Ca²⁺]_i, and the second is assistance of this event by the molecular chaperone protein heat shock protein 90 (35). On competitive inhibition of binding to the caveolae, translocation from the membrane/caveolae to the cytosol with subsequent activation of the enzyme occurs (97). More recent studies have shown the situation to be much more complicated. Several protein kinases have been implicated in eNOS phosphorylation in a variety of cell types, including protein kinase C (77), cAMP-activated protein kinase (12), cGMP-dependent protein kinase II (10) as well as the serine/threonine kinase Akt (21, 29, 30), and the p42 and p44 MAPK, ERK-1/2 (6, 77, 112). The phosphorylated residues are identified as Ser 116 and Thr 497 (495 in human) as well as Ser 1179 for bovine (equivalent to Ser 1177 for human) (28, 30). The role of Ser 116, which is phosphorylated in response to shear stress (30), is unclear at this time. Phosphorylation at position Thr 495, however, lies within the CaM binding region (amino acids 493-511) and apparently blocks eNOS activation (28) that opens many additional regulatory possibilities. Additional studies have also shown that binding to caveolin-1 (a "scaffold" protein located in caveolae) occurs in the same CaM binding region and is also inhibitory (47, 81, 82). In contrast, mutation of Ser 1179 to aspartate, to simulate the negative charge and proper protein conformation observed by phosphate addition, results in a form of eNOS that constitutively produces NO when the mutant cDNA is transfected into cells (21, 29). This phosphorylation event (Ser 1179) appears to be involved in the release of an autoinhibitory loop at the COOH terminus (56). In summary, any new model of eNOS regulation must at the very least include a dynamic interplay between caveolin-1 and eNOS phosphorylation at position 495 competing with Ca²⁺/CaM binding domain to control eNOS together with additional phosphorylation at position 1179. For the purposes of this review, it is also true to say that, regardless of the details, eNOS translocation and activity are both very much dependent on the convergence of cell signaling through Ca²⁺ and protein kinases.

Remapping of cell signaling and differential changes in UA endothelial vasodilator production. With regard to the concept of changes in cell signaling underlying changes in UA endothelial vasodilator production, clearly pregnancy-induced changes in [Ca²⁺]_i vs. ERK signaling would be primary areas with regard to control of cPLA₂ and so PGI₂ production, whereas changes in these same pathways and possibly Akt could be highly significant to general cell responsiveness. A further refinement, however, in any model that arises is the need to explain differential changes in vasodilator production in response to multiple agonists, as reported in our UAEC studies (8, 19). Changes in multiple pathways and a dependence of cPLA₂ and eNOS on distinct kinases may provide one explanation of differential changes in vasodilator production that occur during pregnancy. However, differential regulation of cPLA₂ and eNOS by the same pathways cannot be excluded. The mechanisms that may underlie these changes in cell signaling are now being investigated, and it is no small task because remapping may be achieved through alterations at a number of levels, from heterotrimeric G proteins and/or small G proteins through adapter proteins and protein kinases all the way to cell phosphatases. Recent studies are beginning to unmask these changes for the first time, and the results are not always those expected from studies in other endothelial cells.

Pregnancy-induced changes in UAEC vasodilator production are more clearly associated with changes in kinase activation than with Ca²+ signaling. While the aforementioned studies suggest that changes in UA endothelial function may well be due to changes postreceptor, the picture is complicated still further by the finding in UAEC preparations that not all the agonists that stimulate vasodilator production mobilize Ca²⁺ (8). More specifically, whereas pregnancy-specific activation of NO production is conserved in response to the G protein-coupled receptor agonists ANG II and ATP (19), the ability of ATP to activate phospholipase C is similar in NP-UAEC and P-UAEC. Furthermore, whereas the ability of ATP to mobilize Ca2+ in P-UAEC is more sustained than in NP-UAEC (19; Gifford SM, Cale JM, Tsoi S, Magness RR, and Bird IM, unpublished observations), this difference is not any greater than has been previously reported for hand vein endothelial cells from pregnant and nonpregnant women (75) and so cannot explain the UA-specific change in vasodilator production and associated blood flow. Furthermore, our finding that ANG II has no observable effect on [Ca²⁺]_i levels in P-UAEC or NP-UAEC (8) or in freshly isolated cells (Gifford et al., unpublished observations) confirms that while changes in cell signaling are implicated in pregnancy-induced enhancement of UA endothelial function, changes in Ca²⁺ signaling alone are not sufficient to explain the phenomenon. Further studies have, however, identified pregnancy-specific enhancement of agonist [ATP, ANG II, bradykinin (BK), and growth factors] response coupling to the ERK-1/2 pathway in P-UAEC preparations (8, 19; Gifford et al., unpublished observations), and this clearly involves changes in postreceptor signaling because the same phenomenon is also observed using the receptor independent agonist phorbol ester TPA (8). Recent studies have also confirmed this signaling pathway is activated by the same agonists in freshly isolated UA endothelial cells and is enhanced by pregnancy (Gifford et al., unpublished observations).

Our finding that ANG II did not activate mobilization of Ca²⁺ in UAEC, even though it is associated with a small but significant activation of phospholipase C (8, 19), appeared to be at odds with the previous suggestion that ANG II-sensitive, endothelium-mediated PGI₂ production in UA segments could be blocked by total removal of extracellular Ca²⁺ from the medium or by treatment with verapamil. However, the former experiment using whole vessel segments was measuring endothelial effects by comparing intact with denuded vessels, and there was a significant component of the PGI₂ (measured as 6-keto-PGF₁) from the denuded segment. Whether removal of Ca²⁺ from the medium or treatment with verapamil altered this component is unclear, but it is certainly a concern. Verapamil is a drug that primarily acts on L channels and, unlike VSM cells, endothelial cells at least acutely modulate [Ca²⁺]_i in a verapamil- and nifedipine-insensitive manner (25, 38, 43, 48, 83, 93, 113). Furthermore, treatment of denuded vessels with A23187 significantly increased PGI₂ production in these studies. If we assume, however, the data are not affected by these possible artifacts, our current data showing ANG II can also activate ERK-1/2 in a pregnancy-enhanced manner provides an alternate mechanism for cPLA₂ activation, but raises the question of why removal of Ca²⁺ from the medium would inhibit this response. The current literature on cPLA₂ activation suggests that ERK-1/2 phosphorylation of cPLA₂ sensitizes the enzyme to Ca²⁺ sufficiently so that even resting levels of [Ca²⁺]_i can promote activation, but this means in turn that even small reductions in basal [Ca²⁺]_i may nullify the effect. This is supported by our recent studies of the action of ATP and BK, which both stimulate [Ca²⁺]_i elevation in both NP-UAEC and P-UAEC, but illicit much greater ERK-1/2 activation in P-UAEC than NP-UAEC (8, 19; Gifford et al., unpublished observations). The MAPK/ERK kinase (MEK) inhibitor U0126 blocks ATP stimulation of both NOx and PGI₂ production in P-UAEC, but when [Ca²⁺]_i in UAEC is clamped to below resting levels with BAPTA, the ATP effect on both NO and PGI₂ is again blocked, suggesting even resting Ca²⁺ is critical to both responses. Together these suggest the cell is finely tuned with a resting level of Ca²⁺ just enough to allow ERK activation alone to activate cPLA₂ and eNOS and that even greater effects are seen with further stimulation of Ca²⁺. Thus experimental designs that depress basal [Ca²⁺]_i even slightly may make ERK-1/2 activation insufficient as a stimulus to activate eNOS or cPLA₂. This synergy model has further physiological implications because it suggests agonists that effect ERK (or at least kinase phosphorylation) alone may be greatly potentiated by agents that mobilize Ca²⁺ and likewise agents that mobilize Ca²⁺ may greatly potentiate agents that activate ERK-1/2. The pregnancy-specific shift in UA vasodilation away from PGI₂ toward NO could be explained if the leftward shift in [Ca²⁺]_i sensitivity of eNOS exceeds that of cPLA₂, and our functional data in UAEC suggest that it does (8, 18; Fig. 1). A further implication of this is that the role of growth factors in vivo may be less to control vascular tone directly but to potentiate the effects of Ca²⁺-mobilizing factors released locally, such as ATP. As such, disorders in growth factor secretion and/or action may have a greater effect on pregnancy because of the loss of such synergy.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A and B: effect of altered levels of ERK activation vs. Ca2+ signaling on vasodilator production [endothelial nitric oxide synthase (eNOS) or cPLA2 activation] is outlined for nonpregnant (NP)-uterine artery endothelial cells (UAEC) vs. pregnant (P)-UAEC. C: proposed activity curves for cPLA2 and eNOS across the physiological Ca2+ range in UAEC (80-300 nM). Curves are shown in the absence of agonist-stimulated ERK-1/2 activity (NP-UAEC, broken lines) or presence of ERK-1/2 activation (P-UAEC, solid lines). Based on Refs. 8 and 19.

We now conclude that pregnancy is a time in which there is clearly a dramatic increase in the capacity of UA endothelium to make NO as well as PGI₂, but the actual increase in vasodilator production itself may be more dependent on changes in cell signaling within the UA endothelium and particularly changes in kinases. This leads us to two final questions. 1) How does the remapping of cell signaling occur? 2) How is the change in signaling controlled at the endocrine/physiological level?

CHANGES IN G PROTEINS. As the picture of pregnancy- enhanced vasorelaxation in an NO-mediated fashion was recognized to occur for Ach, BK, and ATP as well as ANG II, it became clear a postreceptor mechanism was possible, but the observation that all these agonist responses are mediated through heterotrimeric G protein-coupled receptors has more recently led to the suggestion that alterations in G protein signaling itself may underlie this adaptive response (110, 120). On the face of it, this proposal has merit and changes in G-subunits may explain in part the enhanced activation of vasodilator production, but this in itself is not likely to fully explain the phenomenon. The same phenomenon of altered NO production responsiveness is observed in response to both bFGF and VEGF in UAEC, yet the involvement of heterotrimeric G proteins is far from clear. Second, the finding that ERK-1/2 activation is increased in response to ANG II, ATP (8, 19), and BK (Gifford et al., unpublished observations) as well as bFGF and VEGF (8) again is at odds with pregnancy-induced changes in UA endothelial cell function being solely due to changes in heterotrimeric G proteins and suggests a mechanism of action at a point downstream (also see below). In addition, the pregnancy-enhanced activation of ERK-1/2 in UAEC is also seen in response to TPA, suggesting again alterations in signaling downstream. More recent data have been presented to support the alteration in G proteins as a mechanism for pregnancy-induced enhancement of UA endothelial function; relaxation of precontracted guinea pig UA is enhanced by NaF (fluoride ions combined with trace amounts of aluminum ions can mimic the terminal phosphate of GTP) in an endothelium-dependent and L-NNA-dependent manner (110). However, although this observation is consistent with the hypothesis, there are problems with this approach. Although the effect of NaF may be both endothelium and NOS dependent, this approach is not specific enough to show its action is at the level of heterotrimeric G proteins alone. It should be noted that other small monomeric G proteins such as Ras are also involved in activation of MEK and therefore for ERK-1/2. Likewise, the further examination shows the effects of ADP-ribosylating factors cholera toxin and pertussis toxin (PTX) (110) assume selectivity of these factors for heterotrimeric G proteins and are endothelium specific when applied to vessel segments. There is mounting evidence to suggest these agents impact on other cell-signaling pathways including the recent report of PTX causing activation of ERK-1/2 in a PKC-dependent but heterotrimeric G protein-independent, Ras-Raf-independent manner (31). Therefore the possible inhibition of G_i may in turn be counterbalanced by activation of MEK in endothelium. Wyckoff and coworkers (124) have used an alternative approach to show the ER in isolated caveoli in fetal pulmonary artery endothelial cells can act through cotransfected G_i but not G_q or G_s to mediate the activation of eNOS via Ca²⁺ and MEK. This strongly supports a potentially important role for G_i in eNOS activation in endothelial cells in general. We also recently identified signals for G_i as well as G_s, G_q, and G_z using cDNA array analysis of mRNA from UAEC (Gifford et al., unpublished observations). There is still much work to be done, however, to prove the proposed hypothesis that changes in heterotrimeric G proteins in the UA endothelium account for changes in functional response to several classes of receptor during pregnancy.

CHANGES IN DOWNSTREAM KINASE SIGNALING. The observation that pregnancy enhances activation of ERK-1/2 in response to both growth factors bFGF and VEGF as well as heptahelical receptors ATP, ANG II, and BK is compelling, but it is also important to recognize that agonist signaling response data from P-UAEC are more similar to those reported in other endothelial cell models such as BAEC or human hand vein endothelial cells (22, 75, 87, 104), whereas that from NP-UAEC is clearly not (8, 19; Gifford et al., unpublished observations). Likewise, we previously commented that the expression levels of eNOS, COX-1, and AT₁-R in UA endothelium from pregnant ewes is actually more similar to that in systemic vasculature, whereas that in UA from nonpregnant ewes is by comparison much lower (9, 45, 71). Thus we can conclude that pregnancy actually changes a poorly responsive UA endothelium to a level of function more like that for many other endothelial cells. This in turn suggests the pregnancy-specific mechanisms present in P-UAEC may not be unique, just lacking or blocked in NP-UAEC.

A detailed examination of current concepts of mechanisms leading to activation of MEK is beyond the scope of this review, but with the use of standard models, the convergence of growth factor and heptahelical receptor signaling based on other cell types would be as described in Fig. 2. It is interesting to note, however, that other mechanisms of activation of eNOS and cPLA₂ must also exist because EGF can also activate ERK-1/2, NO production, and PGI₂ production but with no difference in these responses between NP-UAEC and P-UAEC. That having been said, the primary points of convergence in cell signaling that impact on MEK activation in this proposed model are at the level of Raf (Fig. 2). In our studies, TPA can also differentially activate ERK-1/2, suggesting that differences may occur at the level of PKC. Whether the effects are at the level of expression of PKC isoforms or at the level of targets such as Raf and/or associated proteins is not clear because changes in phosphatases could also cause an alteration in the half-life of protein phosphorylation event. Because many signaling proteins require multiple kinases and often multiple phosphorylation events on a single protein, then the effects could be profound and may explain differential changes in agonist effects on NO vs. PGI₂ production. Whatever the mechanism, our studies are focused on identifying the cause of this pregnancy-specific change in ERK-1/2 activation at this time because we have also found that inhibition of MEK using U0126 blocks both NOx and PGI₂ production in response to ATP (19). This observation is also consistent with the findings of Chen et al. (11) in fetal pulmonary artery endothelial cells that estrogen receptor mediates eNOS activation in an MEK-sensitive manner.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Possible integration of cell signaling pathways as they relate to control of cPLA2 and eNOS. See text for details.

While our attention has been focused on ERK-1/2, it is important not to overlook other potential kinases. Inasmuch as [Ca²⁺]_i is elevated in UAEC by ATP and BK, it is possible CaM kinases may be activated as a result. In addition, we have examined the possible activation of PI-3 kinase/Akt in the control of eNOS. The PI3-kinase antagonist LY294002 does not block activation of eNOS (Ref. 19; Grummer M, unpublished observations) so PI-3 kinase is not the preferred signaling pathway controlling eNOS activity in UAEC. Consistent with this, we have examined the effects of agonists on Akt phosphorylation at amino acid Ser 473. Our data so far suggest that the same agonists that stimulate ERK-1/2 phosphorylation and NO or PGI₂ production do not stimulate a corresponding increase in Ser 473 phospho-Akt. Recent advances now suggest that both Thr 308 and Ser 473 phosphorylation are required for Akt activation, and monitoring Ser 473 phosphorylation alone may not be a clear indicator of Akt activation (37). The question of a role for Akt in the control of eNOS therefore remains unresolved, but our studies to date suggest a role in regulating eNOS is unlikely.

Tonic Regulation vs. Programming of Enhanced UA Endothelial Function

Although future studies will be necessary to conclusively determine what changes in cell signaling are occurring, we can already make some useful general observations on how this may occur. As stated above, pregnancy is associated with changes in steroids, peptide hormones, growth factors, and shear stress. By late pregnancy these have become tonic stimuli to which the UA endothelium is constantly exposed. In addition, the shear stress-induced release of autocrine factors is altered/elevated. Several in vivo models suggest eNOS and, to a lesser extent, COX-1 may well be regulated directly by ovarian and or placental sex steroids (see above). Our recently developed UAEC model supports the notion that eNOS, COX-1, and other UA endothelial proteins are maintained at elevated levels in vivo by tonic stimuli because in all cases the levels decline in culture (8). However, the UAEC model also maintains pregnancy-specific signaling through Ca²⁺ and more specifically the ERK-1/2 pathway seen in freshly isolated cells (Gifford et al., unpublished observations) and the associated ability to make more NOx and PGI₂ in response to agonists previously observed in freshly isolated vessels (8, 19). This must surely indicate pregnancy-specific changes in cell signaling that contribute to increased vasodilator production must occur with some level of independence from tonic stimulation and are probably programmable events. Indeed there may be multiple levels of programming because the change in Ca²⁺ signaling in UAEC is also seen in hand vein endothelial cells, but the change of ERK signaling appears unique to UAEC. The nature of these programming events and molecular mechanisms underlying them are unknown but the recognition of this alone has important implications. This may suggest that such an event must also be actively turned off after termination of pregnancy rather than fade with the loss of a tonic stimulus. In turn, this raises more important questions, namely what the controlling factor is and whether diseases in pregnancy resulting in IUGR/preeclampsia are in fact a failure to program in the first place. This is certainly supported by one of the earliest indications that preeclampsia is associated with a failure to resist the actions of vasoconstrictors, a finding made in early pregnancy before any symptoms of overt hypertension were apparent but later developed (24).

	CONCLUSIONS AND FUTURE DIRECTIONS

TOP ABSTRACT INTRODUCTION IN VIVO PREGNANCY-INDUCED... EX VIVO STUDIES OF... MECHANISMS UNDERLYING CHANGES... CONCLUSIONS AND FUTURE... REFERENCES

Regardless of whether changes in UAEC vasodilator production are due to changes in receptors, kinases, heterotrimeric G proteins, or indeed other molecules, the true significance of advances in this field is the recognition that the mechanisms regulating UA endothelial vasodilator production go far beyond simple receptor-mediated changes in intracellular [Ca²⁺]_i. Furthermore, pregnancy-specific enhancement of UA endothelial function involves reprogramming of cell signaling at the level of protein kinases and/or associated signaling molecules as well as tonic changes in expression of the NO and PGI₂ biosynthetic pathways. It is now clear that at least one major change in UA endothelial function involves alteration of the coupling of multiple receptors to the ERK-1/2 signaling pathway and that this underlies the pregnancy-specific enhancement of PGI₂ production. It is possible this also plays a role in enhancing the activation of eNOS, although alternative or additional roles for other kinases cannot be ruled out. Regardless of the mechanism, it is clear that production of NO and PGI₂ can be differentially regulated to help ensure a successful pregnancy that meets the needs of the fetus without risking the mother. A complete understanding of this event will require studies not only of individual agonist's effects on cell signaling and function but an understanding of how heptahelical receptors, growth factor receptors, and indeed steroid receptors and shear stress act to integrate their signaling to control all aspects of endothelial function. A more complete understanding of these events will in turn provide advances necessary to identify how these critical events may fail and, in severe cases, result in conditions such as IUGR and increased mortality.

In closing we would like to point out once again that other areas of investigation may be equally rewarding. First, the finding that in UAEC the response to EGF does not change as dramatically as does the response to other agonist suggests perhaps a unique role and alternative signaling mechanism for EGF. Second, shear stress is an important physiological regulator of endothelial cell function, but it is not clear how shear stress acts to control UAEC function and whether enhanced responsiveness is due to changes in shear stress or coupling to newly programmed pathways. Third, the concept that changes in cell signaling may underlie changes in UA function may not only apply at the level of endothelium. Although it has been proposed in several studies that PKC is an upstream signal in activation of the ERK pathway in the vascular smooth muscle (49, 50-52, 55, 76), it is noteworthy that a recent study demonstrated a role for ERK in the regulation of PKC as a downstream signal in the UA VSM of pregnant sheep (127). It is clear that pregnancy upregulates the ERK pathway activity in endothelial cells (8, 19) but also in smooth muscle of the UA (127). Thus increased activation of the ERK pathway in UAEC may lead to increased vasodilator release, whereas in UA smooth muscle upregulation of the ERK pathway suppresses PKC activity and decreases uterine vascular tone (8, 19, 127). Although this review finishes with many more questions than it started with, there is clear reason for optimism that significant progress has been made in recent years and we are beginning at last to understand the mechanisms regulating UA function during pregnancy and how their failure may contribute to the development of IUGR.

	ACKNOWLEDGEMENTS

The authors thank J. Sullivan, M. Grummer, and J. Cale for thoughtful discussion of this manuscript.

	FOOTNOTES

This work was supported by National Institutes of Health Grants HL-49210, HL-33255, HL-57653 (to R. R. Magness); HL-57602, HL-64601, USDA 9601773, and 0002159 (to I. M. Bird); HD-38843 (Project I to I. M. Bird, Project II to R. R. Magness); HL-54094 and HL-57787 (to L. Zhang); and HD31226 (Project V to L. Zhang).

Address for reprint requests and other correspondence: I. M. Bird, Univ. of Wisconsin-Madison Medical School, Dept. of Obstetrics and Gynecology, Perinatal Research Laboratories, 7E Meriter Hospital/Park, 202 S. Park St., Madison, WI 53715 (E-mail: imbird{at}wisc.edu).

10.1152/ajpregu.00108.2002

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