Which of the following observations indicates that the placenta has separated?

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Fetal homeostasis depends on the efficiency of the maternal-fetal circulation. As discussed early, maternal arterial pressure propels maternal blood flow through the placenta. The low-resistance uteroplacental vessels accommodate the increased perfusion need for the development of placental vasculature throughout the course of gestation. Therefore, uteroplacental and villous core vasoactivities are essential in maintaining the high-flow maternal blood perfusion into the low-resistance placental intervillous space. Placental vascular reactivity is controlled by several vasodilator and vasoconstrictor systems including the renin-angiotensin system, arachidonic metabolites (thromboxane and prostacyclin), endothelin and its receptors, and NO. Thus, the balance between vasodilators and vasoconstrictors from both the maternal and placental compartments is critical for the homeostatic balance of placental vascular function. Clearly, increased vasoconstriction of the placental vasculature leads to an abnormal course of pregnancy, such as preeclampsia and IUGR. Placental vessels lack autonomic innervation. Hence, humoral effects from the placenta or vasoactivators produced by the placental cells dominate the regulation of fetoplacental vascular reactivity, which is mediated by autocrine and paracrine regulatory mechanisms. Numerous vasoactivators and their corresponding receptors are present in the placenta, and differential synthesis and actions of known vasoactivators are found along the umbilical cord, chorionic plate, and villous vessels () [103].

Figure 8.1

Different actions of vasoconstrictors and vasodilators on umbilical cord and placental vessels. This figure is modified based on reference 102. Ang II: angiotensin II; ET-1: endothelin-1; TXA2: thromboxane A2; BK: bradykinin; 5-HT: 5-hydroxytryptamine; (more...)

8.1. Renin-angiotensin system

The renin-angiotensin system (RAS) or the renin-angiotensin-aldosterone system (RAAS) is an autacoid system that regulates blood pressure, sodium, and fluid homeostasis. When blood volume or Na+ concentration is low, the kidneys secrete renin. Renin cleaves the precursor molecule angiotensinogen, a serum α2-globulin produced in the liver, and converts it to angiotensin I. Human angiotensinogen is 452 amino acids long, and the first 12 amino acids are critically important for its activity. Angiotensin I is 10 amino acids with low biological activity and exists mainly as a precursor to angiotensin II. Angiotensin-converting enzyme (ACE) converts angiotensin I to angiotensin II. ACE is present on all endothelial cells but with highest density in the lung capillaries. Angiotensin II is the major bioactive product of the renin-angiotensin system. In the kidney, angiotensin II has a direct effect on the proximal tubules to increase Na+ reabsorption. By binding to its receptors on mesangial cells, angiotensin II causes these cells to contract along with the blood vessels surrounding them, and promotes the release of aldosterone from the zona glomerulosa in the adrenal cortex to reduce glomerular filtration and renal blood flow and fluid resorption. Angiotensin II is a potent vasoconstrictor that constricts both arteries and veins to increase blood pressure by binding to its receptor AT-1. Angiotensin II has a very short half-life in the circulation, ~30 seconds, while in tissues, its half-life is about 15–30 minutes. Besides angiotensin II {(Ang-(1–8)}, other angiotensin peptides, such as angiotensin III {Ang-(2–8)}, angiotensin IV {Ang-(3–8)}, and angiotensin-(1–7) also have biological activities. For example, angiotensin-(1–7) has become an angiotensin isoform of interest in the past few years, because its cardiovascular and baroreflex actions counteract those of angiotensin II [104]. The metabolic pathway of RAS is shown in .

Figure 8.2

Renin-angiotensin system (RAS) pathway. Renin is produced from prorenin through proteolytic removal of prosegment. The active renin converts angiotensinogen to angiotensin I. Angiotensin I is then converted either to angiotensin II by angiotensin II converting (more...)

During normal pregnancy, maternal RAS components including angiotensin I, angiotensin II and angiotensin 1–7 levels, as well as angiotensinogen and renin activities are all increased compared to the nonpregnant state [105]. Women with uncomplicated pregnancy are normotensive despite a twofold increase in angiotensin II levels and increased circulating blood volume, suggesting that in normal pregnancy the pressor effects of angiotensin II are somewhat compensated [106–108]. Although increased RAS signals with less pressor effect in normal pregnancy is paradoxical, elevated progesterone levels, increased ACE2, and angiotensin 1–7 activities may be the mechanisms for explaining the relative refractoriness to, and less pressor effects by angiotensin II stimulation. Interestingly, normal pregnant women lose pregnancy-acquired vascular refractoriness to angiotensin II within 15–30 minutes after the placenta is delivered [108]. This observation indicates a rapid clearness of substances originating in the placenta. Therefore, placenta-derived progesterone seems a likely candidate for this role [108]. Increased ACE2 and angiotensin 1–7 activities could also be major components counteracting angiotensin II during pregnancy. ACE2 is a carboxypeptidase with 42% homology with ACE, but it exerts different biological activities from ACE. ACE2 generates angiotensin-(1–7) from both angiotensin II and angiotensin I. ACE2 cleaves one amino acid from angiotensin II to form angiotensin-(1–7). ACE2 could also cleave one amino acid from angiotensin I to generate angiotensin-(1–9), which can further be converted to angiotensin-(1–7) by neprilysin and ACE. ACE2 exhibits a high catalytic efficiency to generate angiotensin-(1–7) and at the same time inactivates the vasoconstrictor counterpart angiotensin II [109]. The vasodilatory activity of angiotensin-(1–7) involves the release of nitric oxide, kinins, and prostaglandins. The short half-life of angiotensin II may be explained in part by ACE2 activity in the vasculature. The catalytic activity of ACE2 in generating angiotensin-(1–7) from angiotensin II is about 500-fold greater than that for the conversion of angiotensin I to angiotensin-(1–9) and 10 to 600-fold higher than that of prolyl oligopeptidase and prolyl carboxypeptidase to form angiotensin-(1–7), respectively [110].

A study led by Wallukat discovered that women with preeclampsia have AT-1 agonist autoantibody in their circulation, which is called AT-1-AA [111]. By using sequential amino acid constructs, the same group further found that this autoantibody binds to an amino acid sequence of the second extracellular loop of the AT-1 receptor. Because the PKC inhibitor calphostin prevents the stimulatory effect of this autoantibody, it appears that the AT-1-AA stimulatory effect is PKC-dependent [111]. Since this discovery, several series of experiments have been performed to delineate its contribution to the pathophysiology of preeclampsia. AT-1-AA can cause endothelial cells to express tissue factor, a key enzyme in the extrinsic coagulation cascade [112]. Further support for the role of AT1-AA in preeclampsia comes from the studies showing that AT-1-AA induces oxidative stress including stimulation of NADPH oxidase and activating NF-κB [113]. In addition to oxidative stress and coagulation cascades, AT-1-AA also affects other cellular processes. For example, autoantibody from preeclamptic women is capable of activating angiotensin receptors on placental trophoblasts [114]. The presence of AT-1-AA provides one more potential explanation of the increased vascular reactivity seen in women with preeclampsia.

Fetal placental tissue has its own local RAS system. That is, all components of classical RAS system renin, angiotensinogen, angiotensin I, angiotensin II, angiotensin 1–7, ACE, ACE2, as well as receptors AT-1 and AT-2, have been shown to be present in the fetal placental unit. In 1967, Hodari et al. identified a renin-like substance in human placental tissue [115]. Later, renin, angiotensinogen, ACEs, and angiotensin I and II were all confirmed in the human placenta [116–118]. Kalenge et al. analyzed concentrations of active renin, prorenin, ACE, and angiotensin II in placental tissue and fetal membrane homogenates, and detected all these RAS components in both placental tissues and fetal membranes [117]. They also noticed relative higher values of renin, ACE, and angiotensin II in tissue homogenates from placentas delivered by women with pregnancies complicated with preeclampsia [117]. ACE activity and protein expression are more concentrated in the villous core vessel fraction than in trophoblasts or macrophages [119]. Herse et al. found that messenger RNA expressions for renin, ACE, and angiotensinogen are dominantly expressed in decidua tissues as compared to placental tissues from both normal and preeclamptic pregnant women. In contrast, strong AT-1 receptor expression is found in the placenta verses decidua tissue from both normal and preeclamptic pregnancies [120]. The immunoreactivities for both angiotensin-(1–7) and ACE2 are localized to syncytiotrophoblasts, cytotrophoblasts, villous core fetal vessel endothelium, and vascular smooth muscle cells. Angiotensin-(1–7) expression is reduced in placentas from preeclampsia. We examined AT-1 and AT-2 expressions in first-, second-, and third-trimester placentas and found that in the first-trimester placentas, AT-1 and AT-2 receptors were expressed to both the cyto- and syncytiotrophoblasts. In the second trimester placentas, AT-2 expression was more pronounced in villous stroma and AT-1 was on the apical membrane of syncytiotrophoblasts. In the full-term placentas, strong immunolabeling of these two receptors was observed on villous core fetal vessels. Syncytiotrophoblasts expressed AT-1, but not AT-2 (). The intense AT-1 and AT-2 immunostaining seen in villous core fetal vessel endothelium in third-trimester placentas suggest the role of angiotensin II receptors in relaxation of fetal vessels since activation of AT-1 and AT-2 receptors on endothelial cells results in production of vasodilatory agents, nitric oxide, and prostacyclin (PGI2), which counteract the direct vasoconstrictor effects of angiotensin II on the adjacent smooth muscle cells.

Figure 8.3

Immunostaining of angiotensin II receptors AT-1 and AT-2 in the first-, second-, and third-trimester placentas. A: AT-1; and B: AT-2. 1: First-trimester placentas; 2: Second-trimester placentas; and 3: Third-trimester placentas. Please note the transition (more...)

In preeclampsia, the local placental RAS differs from that in the circulation. By measuring the renin protein and its activity in the supernatants of placental fresh homogenates, Singh et al. found that preeclamptic placentas produced more renin and had elevated renin activity [121]. Higher angiotensin II levels and increased ratio of angiotensin I to angiotensin-(1–7) expression were also found in placental tissues from preeclampsia [122,123]. The presence of AT-1 autoantibody in the preeclamptic placenta further contributes to the altered RAS system in preeclamptic placentas [122]. Therefore, altered placenta RAS system is considered to attribute to the increased vasoconstriction and placental pathophysiology in preeclampsia. Using a unique organ bath perfusion model, we have shown that vasoactivity of chorionic plate arteries from preeclamptic placentas is enhanced compared to that from normal placentas, and that the preeclamptic placenta-derived factors induced chorionic plate artery contraction can be attenuated by AT-1 receptor blocker losartan [124]. These observations indicate that altered RAS pathway metabolites and increased angiotensin II generated within placental tissue contribute to increased vasoconstriction in preeclampsia.

8.2. Arachidonic acid metabolites: thromboxane and prostacyclin

Thromboxane (TXA2) and prostacyclin (PGI2) are members of the family of lipids known as eicosanoids. They are native metabolites of arachidonic acid (5,8,11,14-eicosatetraenoic acid) via the cyclooxygenase pathway. Arachidonic acid is released intracellularly from membrane phospholipids by the enzymes phospholipase A2 and phospholipase C. Microsomal cyclooxygenase subsequently converts arachidonic acid to PGG2, from which PGH2 is formed. Thereafter, three end products, TXA2, PGI2, and stable prostaglandins (PGE2, PGF2α, and PGD2) are generated by their corresponding enzymes, i.e., thromboxane synthase, prostacyclin synthase, and isomerases from PGH2. TXA2 and PGI2 are functional antagonist. TXA2 stimulates platelet aggregation and is a potent vasoconstrictor; In contrast, PGI2 inhibits platelet aggregation and is a potent vasodilator. Unlike other neurotransmitters, such as amines, these arachidonic acid metabolites are not stored, but further metabolized to the end inactive product: TXA2 to TXB2 and PGI2 to 6-keto-PGF1α. The metabolic pathway of TXA2 and PGI2 synthesis is shown in .

Figure 8.4

Arachidonic acid – TX /PGI pathway. Phospholiase A2 (PLA2) and phospholipase C (PLC) liberate arachidonic acid (AA) from membrane lipid of placental syncytiotrophoblasts. Cycloxygenase (COX) produces PGG2 and PGH2 from AA. Subsequently, thromboxane (more...)

TXA2 was initially described by Palmer et al. as a rabbit aorta-contracting substance [125], which has a potent contractile potency toward vascular smooth muscle cells [126]. TXA2 is mainly produced by activated platelets and has prothrombotic properties, stimulating activation of new platelets, as well as increasing platelet aggregation. Platelets are the main source of TXA2 in the circulation. The molecular weight of TXA is 60kDa. TXA2 has a very short half-life of about 30 seconds, and it is rapidly hydrolyzed to the biologically inactive TXB2, which is stable and often used as an indicator of TXA2 production/level in biological samples. When TXA2 is produced from PGH2 by thromboxane synthase, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA) are also simultaneously produced [127].

Because of its short half-life, TXA2 primarily functions as an autocrine or paracrine mediator by binding to its receptor, thromboxane receptor (TP receptor), in the nearby tissues such as on platelets and endothelial cells surrounding its site of production. TP receptor is a G-protein coupled receptor. The gene for the TP receptor is located on chromosome 19. Two TP receptor isoforms have been found [128]. One was cloned from human placenta and the other from human endothelial cells. The former was referred as TP alpha and the latter as TP beta. Both isoforms are present in platelets [128]. They differ only in their carboxyl-terminal tails, but their function is different. TP alpha activates adenylyl cyclase, while TP beta inhibits it. Different G-protein receptor activation accounts for the downstream effects of TP-receptor responses. In fact, early work has shown that TP- mediated platelet shape changes are mainly dependent on G12/13 activation, while Gq activation is responsible for platelet aggregation [129]. The mechanism of TXA2 elicited vascular smooth muscle constriction is involved in Ca2+ signaling. TP receptor activation causes phospholipase C-catalyzed phosphoinositide hydrolysis, which in turn mobilizes intracellular Ca2+ and Gq acts as a trimeric G protein coupled to TP receptor to activate phospholipase C [130].

Prostacyclin was discovered by Moncada et al. in 1976 [131]. They identified a lipid mediator that could inhibit platelet aggregation and they called it “PG-X,” [131]. PG-X later became known as PGI2. It is 30 times more potent than any other known antiaggregatory agent [131]. PGI2 is mainly produced by vascular endothelia cells. PGI2 is a potent platelet inhibitor and a strong vasodilator. It counteracts with TXA2 and plays a key role in maintenance of cardiovascular homeostasis related to vascular damage. Similar to TXA2, PGI2 also achieves its function through a paracrine and autocrine signaling cascade by binding to G-protein coupled prostacyclin receptor (IP receptor) on target cells including platelets and endothelial cells. To inhibit platelet aggregation, PGI2 binds to IP receptor on platelets. The receptor activating process turns on adenyly cyclase to produce cAMP, which inhibits platelet activation and aggregation to counteract increased cytosolic calcium levels induced by TXA2. To inhibit vasoconstriction, PGI2 binds to IP receptors on vascular smooth muscle cells. Receptor activation turns on adenyly cyclase to increase cAMP levels in the cytosol. cAMP then activates protein kinase A (PKA), which continues the cascade by phosphorylating and inhibiting myosin light-chain kinase, which leads to smooth muscle relaxation and consequently vasodilatation.

In addition to arachidonic acid, 5,8,11,14,17-eicosatetraenoic acid (EPA) is also a substrate for thromboxane synthase and prostacyclin synthase. EPA produces TXA3 and PGI3. TXA3 has less effect on platelet aggregation than TXA2, but PGI3 has equal activity as PGI2 in inhibiting platelet aggregation and promoting vasodilatation.

Placental trophoblasts are a particularly rich source in the cyclooxygenase substrate arachidonic acid. Arachidonic acid is involved in cell membrane biosynthesis and represents a large portion of the total syncytiotrophoblast lipid [132]. Syncytiotrophoblasts are capable of incorporating arachidonic acid into both the brush border membrane (BBM) and basal plasma membrane compartments [133]. Trophoblast cells produce both TXA2 and PGI2. Placenta production of prostacyclin-like substances was first described by Myatt and Elder in 1977 [134]. They found a substance produced by human placenta that was capable of inhibiting platelet aggregation and discovered that this substance was heat sensitive [134]. Later, it was found that PGI2 production (measured by its stable metabolite 6-keto-PGF1α), was significantly reduced in placentas delivered by women complicated with preeclampsia [135]. In contrast, production of TXA2 was significantly increased [136,137]. The imbalance of increased TXA2 production and decreased PGI2 production in the preeclamptic placenta is believed to play a significant role in inducing placenta vasoconstriction in preeclampsia. The reason for altered TXA2 production in placentas is not clear, but oxidative stress is considered one of the possible mechanisms that regulate TXA2 and PGI2 production. Study has shown that hypoxia not only promotes TXA2 and PGI2 productions, but also increases phospholipase A2 production [138], which indicates that increased phospholipase A2 release/activity can liberate further arachidonic acid from trophoblast membrane phospholipids to promote TX production. This results in an imbalance of increased TXA2 and decreased PGI2 production, consequently driving vasoconstriction of placental vessels in preeclampsia [138].

Consistent with the altered TXA2 and PGI2 productions in preeclamptic placentas, the ratio of TXA2 levels and PGI2 levels in maternal circulation are also increased in women with preeclampsia, and this increase is linked to the severity of disease [139]. The evidence of imbalanced thromboxane and prostacyclin in both the maternal circulation and the placenta compartment provides the rationale for the “low-dose aspirin therapy” for prevention of preeclampsia [140,141]. Aspirin is a nonsteroidal antiinflammatory drug and a nonselective inhibitor of cyclooxygenase. Aspirin inhibits both the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isoenzymes. By inhibiting the COX enzyme to limit the production of the precursor of thromboxane within platelets, aspirin irreversibly blocks the formation of TXA2 in target cells and consequently inhibits platelet aggregation. This anticoagulant property makes aspirin useful for reducing the incidence of cardiovascular diseases including heart attacks. Although results from low-dose aspirin therapy clinical trials in preventing preeclampsia are inconsistent, timing and dose of the aspirin used in the various clinical trials and the heterogeneity of preeclampsia, early versus late onset, and/or with or without other clinical complications, may also contribute to the unsatisfactory outcomes [142]. Nonetheless, altered arachidonic acid-cycloxygenase-thromboxane pathway regulation in the placenta is widely believed to play an important role in the pathophysiology of preeclampsia.

We examined PGIS, TXS, and their receptors IP and TP expressions in placental tissues at different gestational ages in normal pregnancy and found that PGIS is strongly expressed in both cyto- and syncytiotrophoblasts in the first-trimester placenta. PGIS expression is reduced in advanced gestation (). TXS is expressed in syncytiotrophoblasts throughout pregnancy. Both PGIS and TXS are expressed in the villous core fetal vessel endothelium in third-trimester placentas. Compared to TP receptor, IP receptor is strongly expressed in trophoblasts and fetal vessels in the first- and second-trimester placentas. Fetal vessel endothelium expresses both IP and TP receptors in the third-trimester placentas (). The pattern for IP receptor over TP receptor expressions in trophoblasts and villous core vessels suggests that PGI2 mediated vasodilatory activity is dominant in the placenta during normal pregnancy.

Figure 8.5

Immunostaining of prostacyclin synthase (PGIS), IP receptor, thromboxane synthase (TXS), and TP receptor in first-, second-, and third-trimester placentas. A: PGIS; B: IP receptor; C: TXS; and D: TP receptor. 1: First-trimester placentas; 2: second-trimester (more...)

Other than the anticoagulation and vasodilation properties of PGI2, recent studies also show that PGI2 plays a role in angiogenesis, and is involved in proinflammatory and/or antiinflammatory responses in endothelial cells. For instance, an in vitro study has shown that prostacyclin analogs could stimulate VEGF production by human lung mesenchymal cells/fibroblasts [143]. Kamio et al. studied prostacyclin effects on human lung fibroblasts using the prostacyclin analogs iloprost and beraprost [143], and found increased VEGF mRNA expression and protein release in cells treated with iloprost and beraprost. This prostacyclin-analog induced VEGF expression and production could be blocked by the adenylate cyclase inhibitor SQ-22536 and by a protein kinase A (PKA) inhibitor KT-5720 [143], indicating that prostacyclin downstream effects on VEGF is mediated via cAMP-activated PKA signaling cascade [143]. The finding of colocalizatioon of PGI2 synthase with caveolin-1 in endothelial cells also suggests a potential angiogenic function of PGI2 [144]. It is known that caveolin-1 expression is critical for VEGF-induced angiogenesis [145]. Both cytotrophoblasts and syncytiotrophoblast express caveolin-1 [146] and PGI2 synthase and IP receptor are strongly expressed in the first-trimester placental cytotrophoblasts (). It is highly likely that cytotrophoblasts-derived angiogenic factors are fundamental to stimulating villous core stromal vasculogenesis. The positive staining of PGI2 synthase in the villous stromal cells [147] also suggests that prostacyclin derived from stromal mesenchymal cells may play a role in a bidirectional signaling network between the mesenchymal and vascular cells to promote vasculogenesis and angiogenesis during placenta development.

8.3. Endothelin-1 and its receptors

Yanagisawa et al. found that a peptide originally derived from the supernatant from porcine aortic endothelial cells has a potent vasoconstrictive effect on porcine coronary artery strips [148]. The peptide was then purified and cloned from endothelial cells and named endothelin because it was derived from endothelial cells [148]. Endothelin has three isoforms, endothelin-1, -2, and -3. Endothelin-1 (ET-1) is the most potent and long lasting vasoconstrictor known, being 100 times more potent than noradrenaline [148]. Mature ET-1 is a 21-amino acid peptide, and it is a main member of the endothelin peptide family [149]. Endothelins has two receptors, ETA and ETB. ET-1 and -2 bind to ETA and ETB, while ET-3 only binds to ETB. ETs have been demonstrated to play important roles in cardiovascular diseases including hypertension, atherosclerosis, diabetes, and renal diseases [150].

In the full-term human placenta, the immunoreactivity of ET-1 is localized to endothelial cells of capillaries of placental microvilli, small- and medium-sized arteries and veins as well as placental syncytiotrophoblasts [151,152]. Studies also confirmed a broad distribution of ET-1 expression in placentas throughout gestation in which ET-1 expression is increased along with gestational age through the first trimester to full term [153]. Trophoblasts produce/release ET-1 [152,154]. Human placenta also expresses ETA and ETB receptors. We found that ETB is strongly expressed in placental trophoblasts and its expression is increased along with gestational age (). In contrast, ETA is weakly expressed in placental trophoblasts. Although the reason for the differential expression of ETA and ETB in placental trophoblasts is not clear, an in vitro binding assay has clearly shown that ET is capable of binding to the isolated trophoblast membranes [155]. ET-1 is also involved in trophoblast invasion and differentiation of trophoblast cells isolated from first-trimester placentas [156].

Figure 8.6

Immunostaining of ETA and ETB in first-, second-, and third-trimester placentas. A: ETA; and B: ETB. 1: First-trimester placentas; 2: second-trimester placentas; and 3: third-trimester placentas. ETB is strongly expressed in placental trophoblasts and (more...)

The potential role of placenta-derived ET-1 in placental vessel vasoconstriction was studied in a vessel ring organ bath perfusion model [124]. It was found that conditioned medium derived from tissue culture of preeclamptic placentas could induce constriction of the chorionic plate artery ring of placentas from noncomplicated pregnancies and this vasoconstrictive effect could be attenuated by both ETA and ETB receptor antagonists [124], suggesting that ETA and ETB receptors are present in chorionic plate vessels and ET-1 produced by trophoblasts could induce vasoconstriction in the placenta [124]. Consistent with our organ bath perfusion study, Wilkes et al., also found that ET-1 could induce a sustained dose-dependent increase in perfusion pressure in umbilical cord artery and vein [157]. Paradoxically, ET-1 gene expression found no difference between placentas from normal and preeclamptic pregnancies [158]. Although the reason for this is not clear, the density difference between ETA and ETB receptors on placental trophoblasts could be an explanation. Activation of ETA receptors on vascular smooth muscle cells induces vasoconstriction and activation of ETB receptors on endothelial cells induces vasodilatation. Down-regulation of ETA receptor expression but relatively increased ETB receptor expression were found in preeclamptic placentas [158,159]. The increased ETB receptor expression could be a compensatory effect that accounts for the partial protective effect during preeclampsia [158,159].The protective effect of ET-1 mediated by ETB receptors is further supported by a study showing that ET-1 attenuated apoptosis of trophoblasts from full-term human placentas [160]. However, functions other than vasoactivity of ETs and their receptors are largely unknown in trophoblasts.

8.4. Nitric oxide

Nitric oxide (NO) synthesis is catalyzed by nitric oxide synthase (NOS), which converts L-arginine to L-citruline with NO as a free radical by-product. There are three NOS isoforms, iNOS, eNOS, and nNOS, and the predominant constitutive NOS isoform within the placenta is eNOS. The biological effect of NO is mediated by two pathways: (1) the cGMP-dependent pathway involves activation of the NO-sensitive soluble form of guanylyl cyclase (sGC). This enzyme is responsible for cyclic-guanosine-monophosphate (cGMP) generation and protein kinase-G (PKG) activation; and (2) the cGMP-independent pathway involves the signaling molecules such as Ca2+-activated and ATP-activated K+ channels. Protein S-nitrosylation has been suggested as the possible underlying mechanism [161,162].

NO plays an important role, along with other vasodilators PGI2, natriuretic peptides, and endothelial-derived hyperpolarizing factor (EDHF), in uteroplacental vascular adaptation during pregnancy. Strong eNOS activity is found in the vascular endothelium of the umbilical cord, chorionic plate, and stem villous vessels. De novo NO produced by the endothelium is secreted adluminally to adjacent vascular smooth muscle cells (). The adluminally directional release of NO supports the role of NO in the transformation of the uterine arteries and favors its participation in maintaining the uteroplacental vasorelaxation.

Figure 8.7

NO pathway. The constitutive nitric oxide synthase (eNOS) is expressed in vessel endothelium. When acting on its substrate L-arginine, nitric oxide (NO) is produced as a by-product to generate L-Citruline. NO diffuses to the underlying vascular smooth (more...)

The importance of NO is highlighted by the improvement of the local and systemic perfusion changes in hypertensive pregnant patients supplemented with L-arginine. In patients with mild preeclampsia, administration of NO donor glyceryl trinitrate significantly reduces the blood flow resistance in fetoplacental circulation as examined by Doppler monitoring [163]. Consistently, inhibition of NOS activity on isolated human stem villous arterioles increases umbilical vascular resistance with reduced blood flow [164]. Additional evidence comes from the experiments showing that inhibition of sGC increases the perfusion pressure of the human fetoplacental circulation [165]. Substantial evidence from animal studies has also shown a key role of NO in shear stress mediated vasodilation in the uteroplacental vasculature [166]. Thus, it is clear that NO is an important endogenous dilator of the fetal vessels in the placenta.

Several studies have found that eNOS activity and NO end-products (nitrites and nitrates) are significantly high in villous homogenate and eNOS is intensively expressed in endothelium of stem villous vessels and small arteries of terminal villi in preeclamptic placentas compared to those in normal placentas [167,168]. These observations suggest that increased placental NO production may represent a compensatory mechanism to offset the pathologic effects in preeclampsia. In other studies, L-arginine levels, a substrate for endogenous NO synthesis in umbilical blood and in villous tissues, were found lower, but nitrotyrosine staining, a marker of peroxynitrite, was stronger in preeclamptic than in normotensive pregnancy [169]. Gene expression and protein tissue content of arginase II, the enzyme that degrades arginine to ornithine, were also found higher in preeclamptic placentas than in normotensive pregnant placentas [169]. Although it sounds controversial, it is clear that in the normal placenta, adequate L-arginine/NOS pathway regulation orients eNOS toward NO, whereas in preeclampsia a lower than normal L-arginine level induced by arginase II overexpression may redirect eNOS toward peroxynitrite formation and contribute to vasoconstriction in preeclampsia [169].

eNOS activity has also been described in trophoblasts of early trimester placentas. Immunohistochemistry study of the first-trimester placenta demonstrates the presence of eNOS in the cell columns of anchoring villi and in extravillous trophoblasts at the implantation site and in villous syncytiotrophoblasts, indicating that in situ produced NO by trophoblasts may participate the relaxation of vascular wall at the implantation site. In addition, the observation of high levels of eNOS immunoreactivity in the intermediate trophoblasts in complete hydatidiform mole and in choriocarcinoma compared to placentas with nontrophoblast disease suggests that eNOS is also capable of stimulating trophoblast proliferation [170,171].

Recently, two eNOS regulating proteins, an eNOS traffic inducer (NOSTRIN) and an eNOS-interacting protein (NOSIP) were reported [172]. NOSTRIN is found to colocalize extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells, and eNOS-NOSTRIN interactions are confirmed in both in vitro and in vivo experimental studies [172]. Furthermore, NOSTRIN overexpression could induce a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release [172]. These data indicate that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS. Although, at present, no information is available regarding NOSIP and/or NOSTRIN in placental trophoblasts or villous core vessels, it is expected that NOSIP and NOSTRIN may regulate eNOS translocation and trafficking in placenta vasculature.

8.5. Chymase

Chymase is a chymotrypsin-like serine protease. Chymase has no enzymatic activity in the normal state but is activated immediately upon release into the extracellular matrix. Chymase activation is associated with many pathophysiological conditions and plays an important role in vessel reactivity because it is involved in both angiotensin II and endothelin biosynthesis. Like ACE, chymase converts angiotensin I to angiotensin II by hydrolyzing bonds between Phe8-His9. Chymase is considered a potent non-ACE angiotensin II generating enzyme and found to be responsible for approximately 70–80% angiotensin II generated in the human heart tissue [173]. Chymase is also an endothelin-forming enzyme. It cleaves the Trp21-Val22 bond of big-ET-1(1–39) forming ET-1(1–21) and the Tyr31-Gly32 bond, resulting in the formation of ET-1(1–31) [174]. Both ET-1(1–21) and ET-1(1–31) bind to ET-A receptor on vascular smooth muscle cells. Angiotensin II and ET-1 are potent vasoconstrictors, thus chymase plays a fundamental role in hypertension and atherosclerosis. The major pathophysiological effects of chymase are shown in .

Figure 8.8

Potential role of chymase in the cardiovascular system.

The chymase gene is found in the placental trophoblasts and the open reading frame of the chymase gene in trophoblasts is 100% homologous to that reported in the human heart tissue and mast cells [175–177]. Importantly, chymase expression is up-regulated and chymase activity is increased in trophoblasts of placentas from women with preeclampsia [175], suggesting that chymase may contribute to the altered RAS system, as well as to ET-1 production in preeclamptic placentas. The placental chymase is mainly localized in syncytiotrophoblasts [175]. The evidence of chymostatin- blocking effects on preeclamptic placenta-derived factor induced vasoconstriction of chorionic plate arteries and placental vessel smooth muscle cells further indicates the role of trophoblast- derived chymase mediated placenta vasoactivity in preeclampsia [124,178].

In addition to vasoconstrictor generating activity, chymase also plays a role in atherosclerosis by degrading HDL and inhibiting the apolipoprotein-mediated removal of cholesterol [179,180]. Chymase is considered to be an inflammatory protease [181]. It triggers the production of cytokines and chemokines, which stimulate the infiltration of inflammatory cells. In mast cells, degranulation not only release chymase but also biogenic amines, TNFα, and serglycin proteoglycans, as well as various lysosomal enzymes [182]. Similarly, increased chymase activity is associated with endothelial activation and increased endothelial adhesion molecule expression [183]. Although the inflammatory activity of chymase is not clear in the placenta, it is expected that the role of chymase in the placenta is beyond the ACE and ECE properties, since the sheddase activity of chymase was found to be associated with increased soluble VEGF receptor-1 (sFlt-1 release) in preeclamptic placentas [184].

8.6. Role of trophoblasts in regulation of placental vasoactivity

The syncytio layer of trophoblasts has a unique hemonochorial structure: the apical surface of trophoblasts bathes in the maternal blood in the intervillous space, while the basal membrane (BM) is close to the villous stroma and the fetal vasculature. This anatomic feature of placental trophoblasts makes them an ideal candidate to mediate the signal from the maternal source and to regulate the function of underlying tissues, including villous core fetal vessels. Placental trophoblasts are polarized epithelial cells. These cells have diverse properties in chemical and biochemical compositions between the apical surface (also called microvillous membrane/MVM or brush border) and the basal membrane [185]. The brush border is enriched with membrane-bound alkaline phosphatase and 5' nucleotidase, while the basal side is enriched with Na+-K+ ATPase and F(-)-stimulated adenylate cyclase [186]. Compelling evidence has also shown differential characteristics of MVM from BM in transporting nutrients across the placental barrier from the mother to fetus [187]. It has also been demonstrated that >90% of matrix metalloproteinase-2 (MMP-2) and MMP-9 produced by trophoblasts are secreted into the basolateral direction. [188].

In spite of the aforementioned evidence about trophoblast polarity, little information is available regarding the possible polarized secretion of vasoactivators from placental syncytiotrophoblasts. However, several lines of evidence highlight this possibility. The placenta is devoid of a nervous system. Thus, it must depend on locally produced factors to regulate the vasoactivity within the placenta. This notion is best illustrated in the case of preeclampsia. Preeclampsia is characterized by systemic vasoconstriction. In particular, the uteroplacental vasculature is prominently affected. The underlying mechanism has been attributed to the imbalance of increased vasoconstrictors and decreased vasodilators exemplified by TXA2 and PGI2. What seems missing is an explanation for how the short-lived TXA2 secreted from the placental trophoblasts could mediate constriction of placental vessels. Chorionic arteries from preeclamptic placentas displayed a greater contractility than that from normal placentas either to the vasoconstricting agent KCL or placenta conditioned medium [124], which were corroborated by corresponding inhibitors to thromboxane, endothelin, and angiotensin II/chymase. Based on the anatomical reciprocation between placental syncytiotrophoblasts and placental vessels, it is logical to reason that trophoblasts play a significant role in affecting vasoactivity in the placenta.

We recently found that predominant basal directional release of vasoconstrictor(s) is very likely a feature of syncytiotrophoblasts in regulation of placental villous core vessel activity during pregnancy, especially in preeclampsia [147]. TXS expression is increased in placental trophoblasts in preeclampsia [147]. Trophoblasts release TXA2 to both the apical and basal directions, and the bidirectional release of TXA2 is increased in trophoblasts from preeclamptic placentas [147]. Most interestingly, apical exposure of trophoblasts to arachidonic acid, a substrate of cyclooxygenase, results in an increase in TXA2, but not PGI2, releases in both the apical and basal directions. However, apical exposure of cyclooxygenase inhibitor aspirin could only inhibit the bidirectional release of TXA2 in trophoblasts from normal placentas but not in trophoblasts from preeclamptic placentas. These observations suggest that arachidonic acid induced increase in bidirectional release of TXA2 is mediated via cyclooxygenase in the normal trophoblasts, whereas TXS probably plays a dominant role in producing TXA2 and regulating the bidirectional release of TXA2 in the preeclamptic placenta, since upregulation of TXS is seen in preeclamptic placentas [147].

Although at the present time, only thromboxane has been shown to have a polarized secretion pattern from the placental syncytiotrophoblasts, it can be anticipated that other vasoactivators might also present a similar pattern. Chymase expression is upregulated in placental trophoblasts in preeclampsia [175]. An in vitro study has shown that chymotrypsin/chymase promotes basal directional release of angiotensin II by endothelial cells [189]. In the endothelium, ET-1 is also predominantly released abluminally toward the vascular smooth muscle, suggesting a paracrine role in the regulation of vascular smooth muscle contraction [190,191]. Chymase acts as both ACE and ECE to promote angiotensin II and ET-1 productions. Therefore, increased trophoblast chymase activity is likely to contribute to the increased placental vasoconstriction mediated by basal directional release of both angiotensin II and ET-1 in preeclampsia.

What are the signs of separation of placenta?

Which observation would suggest that placental separation is occurring?

What happens when the placenta separates?