INTRODUCTION

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

NORMAL PREGNANCY IS ASSOCIATED with reductions in vascular resistance and arterial pressure. However, in 5-10% of pregnancies in the US and 15% of pregnancies among African-Americans, women may have hypertension as one complication of pregnancy (101, 143). Hypertension in pregnancy is related to one of four conditions: chronic hypertension that predates pregnancy; preeclampsia-eclampsia; chronic hypertension with superimposed preeclampsia; and gestational hypertension, a nonproteinuric hypertension of pregnancy (20, 172). Preeclampsia is a serious, systemic syndrome of elevated blood pressure, proteinuria, and other clinical findings. Although preeclampsia is a major cause of maternal and fetal morbidity and mortality, the exact mechanisms of this disorder have not been clearly identified. Understanding the mechanisms of preeclampsia should help develop new strategies for prevention and treatment of this disorder. Because preeclampsia is a disease of humans, clinical studies in hypertensive pregnant women and on samples from their plasma, body fluids, and postpartum placentas have been very useful in identifying the possible mechanisms of the disease. Several excellent reviews have provided detailed information regarding the general pathophysiology and the clinical aspects of preeclampsia, and the reader is encouraged to refer to some of them (10, 53, 66, 67, 134). However, investigation of the cellular and molecular mechanisms of hypertension in pregnant women could be difficult and costly. This led investigators to perform experimental studies in animal models of hypertension in pregnancy. Although the terminology may not be completely accurate, for the sake of clarity and to avoid confusion with preeclampsia in human pregnancy, we will refer to the hypertension in pregnant animal models as pregnancy-induced hypertension (PIH). Several prior reviews highlighted the significant changes in renal control mechanisms of arterial pressure in animal models of PIH and the alterations in kidney functions as possible causes of the increased arterial pressure in preeclampsia (75, 76, 102). However, hypertension is a multifactorial disorder that could involve additional alterations in the vascular and neurohumoral control mechanisms of the arterial pressure.

The purpose of this review is to make use of data largely derived from animal models of PIH to provide insight into the possible vascular and cellular mechanisms of the increased arterial pressure in preeclampsia. In this review, some of the hemodynamic changes that occur during normal pregnancy and preeclampsia will first be outlined. The possible initiating events that could trigger the development of preeclampsia will then be briefly described. We will follow with a detailed description of the intermediary changes in the endothelium-dependent mechanisms of vascular relaxation and the mechanisms of vascular smooth muscle contraction and how these vascular changes might relate to the increases in vascular resistance and arterial pressure as observed in women with preeclampsia and in animal models of PIH. The review will end with a perspective on potential areas for future investigations to better understand the vascular mechanisms of the increased arterial pressure in preeclampsia.

	HEMODYNAMIC AND VASCULAR CHANGES DURING NORMAL PREGNANCY AND PREECLAMPSIA

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

Normal pregnancy is associated with significant hemodynamic and cardiovascular changes to meet the metabolic needs of the mother and fetus. For example, the maternal cardiac output and plasma volume increase during pregnancy, whereas the total vascular resistance and arterial pressure tend to decrease (139). Also, normal pregnancy is associated with increased renal plasma flow, decreased renal vascular resistance, and decreased pressor response and vascular reactivity to vasoconstrictors such as -adrenergic agonists and ANG II (26, 39, 44, 48, 56, 70, 91, 112).

Although a hyperdynamic circulation may occur before the clinical onset of preeclampsia (57), the clinical phase of the disease is associated with severe increases in vascular resistance and arterial pressure, enhanced pressor response to vasoconstrictors such as ANG II, and reduction in renal plasma flow (103). The triggering mechanisms that lead to the dramatic hemodynamic and vascular changes observed during preeclampsia have been very elusive; however, most investigations have centered on a possible role of the placenta.

	PLACENTAL ISCHEMIA AS AN INITIATING EVENT OF PREECLAMPSIA

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

Preeclampsia develops during pregnancy and remits after delivery, implicating the placenta as a central culprit in the disease. During the early stages of normal pregnancy, the cytotrophoblasts invade the uterine spiral arteries and progressively replace the vascular endothelial cells, the medial elastic tissue, the smooth muscle layer, and the neural tissue. By the end of the second trimester, the spiral arteries are turned into dilated tubes lined by cytotrophoblast. This remodeling of the uterine spiral arteries results in the formation of a low-resistance arterial system, which ensures sufficient blood supply and nutrition to the growing fetus.

In preeclampsia, abnormal expression of the adhesion molecule integrins by the cytotrophoblasts as well as widespread apoptosis of invasive cytotrophoblasts leads to limited invasion of the uterine spiral arteries to only the superficial layers of the deciduas (54, 71, 173). The shallow cytotrophoblast invasion of the deciduas and the inadequate vascular remodeling of the uterine spiral arteries does not meet the fetal blood flow and nutrition demands and may lead to intrauterine growth retardation, a common observation during preeclampsia. In addition to its deleterious effects on the growing fetus, placental ischemia could also initiate a cascade of events leading to dramatic changes in the maternal circulation during preeclampsia.

Because of the difficulty of performing mechanistic studies in pregnant women, several animal models of PIH have been developed to test the role of placental ischemia as a possible initiating event of the elevated arterial pressure during preeclampsia (4, 6, 27, 38, 58, 105). Although experimental induction of chronic uteroplacental ischemia in pregnant animals has shown variable effects in different species and preparations (127), it is considered one of the promising animal models of PIH. Studies in late pregnant sheep, dog, and rabbit showed that reduction in uteroplacental perfusion pressure induces a hypertensive state that resembles hypertension in human pregnancy and provided evidence for a possible relationship between placental ischemia and preeclampsia (27, 58, 105, 127). However, the intermediary mechanisms between placental ischemia and the increased arterial pressure in human preeclampsia and animal models of PIH are not clearly understood. Recent studies in a rat model of reduced uterine perfusion pressure (RUPP) produced by clipping the lower abdominal aorta and the main uterine branches of both the ovarian arteries during late pregnancy provided evidence for significant changes in renal functions as possible causes of the increased arterial pressure in this animal model of PIH (4, 6), and these studies have previously been discussed in prior reviews (75, 76). Other studies focused on the possible vascular and cellular mechanisms of the increased arterial pressure in the RUPP rats and the mechanisms by which a localized reduction in uteroplacental perfusion pressure during late pregnancy could cause generalized increase in vascular reactivity and thus lead to increased vascular resistance and arterial pressure (38).

	ENHANCED VASCULAR REACTIVITY IN PREECLAMPSIA

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During normal pregnancy the pressor response to vasoconstrictor agonists appears to be reduced (26, 56, 70, 112). Also, the vascular reactivity to vasoconstrictor agonists such as the ₁-adrenergic agonist phenylephrine (Phe) and ANG II is reduced in pregnant rats compared with virgin rats (39, 44, 91, 112). In contrast, preeclampsia is characterized by generalized vasoconstriction and increased pressor response to vasoconstrictor agonists such as ANG II (103). The increased vascular reactivity to vasoconstrictors during preeclampsia could be due to decreased endothelium-dependent mechanisms of vascular relaxation and/or enhanced mechanisms of vascular smooth muscle contraction.

	ENDOTHELIAL CELL DYSFUNCTION DURING PREECLAMPSIA

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

The decreased vasopressor responses and vascular reactivity to vasoconstrictor agonists during normal pregnancy have been attributed, in part, to increased synthesis/release of nitric oxide (NO) and perhaps other vasodilator substances such as prostacyclin (PGI₂) and hyperpolarizing factor (EDHF) by various maternal cells including vascular endothelial cells (Fig. 1) (2, 16, 17, 32, 68, 72, 125, 151, 165, 170). This led to the hypothesis that preeclampsia is an endothelial cell disorder and that the increased vascular resistance and arterial pressure during preeclampsia are possibly due to endothelial cell dysfunction and alterations in endothelium-dependent vascular relaxation (66, 114, 138).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Vascular changes during normal pregnancy and preeclampsia. During normal pregnancy there is an increase in the activity of endothelial nitric oxide synthase (NOS) and cyclooxygenase (COX) and increased production of nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF). NO increases cGMP and PGI2 increases cAMP in smooth muscle, which decrease intracellular Ca2+ and the myofilament sensitivity to Ca2+. Also, EDHF opens K+ channels in smooth muscle, leading to membrane hyperpolarization. This leads to smooth muscle relaxation and decreased peripheral resistance and arterial pressure. In preeclampsia there is increased release of placental cytokines that inhibit the production of endothelium-derived relaxing factors and thereby decrease smooth muscle relaxation. Cytokines also stimulate the release of endothelium-derived contracting factors such as endothelin (ET-1) and thromboxane (TXA2) and could activate the renin-angiotensin system (RAS) in the kidney leading to increased ANG II. ET-1, TXA2, and ANG II stimulate specific receptors in smooth muscle leading to increased intracellular Ca2+, protein kinase C (PKC) activity, smooth muscle contraction, and increased peripheral resistance and arterial pressure. ER, endoplasmic reticulum; SR, sarcoplasmic reticulum.

There is ample clinical and biochemical evidence of endothelial cell dysfunction during preeclampsia (137). Studies in women with overt preeclampsia showed increases in circulating levels of cellular fibronectin and factor VIII-related antigen, both of which are markers of endothelial cell injury (65, 138, 140). The increased levels of these markers precede clinically overt preeclampsia and disappear with resolution of the disease, providing evidence for a possible causal relationship between endothelial cell injury and preeclampsia (135).

We recently used the RUPP rat model of PIH to test the hypothesis that localized reduction in uterine perfusion pressure during late pregnancy is associated with enhanced systemic vascular reactivity and impaired endothelium-dependent vascular relaxation (38). We found that the reactivity of endothelium-intact vascular strips to Phe is enhanced in RUPP rats compared with normal pregnant rats. Removal of the endothelium significantly enhances the Phe contraction in pregnant rats, but to a lesser extent in RUPP rats (Fig. 2). Also, the ACh-induced relaxation is less in RUPP rats than normal pregnant rats. These studies suggested that an endothelium-dependent relaxation pathway is intact in pregnant rats but is impaired in RUPP rats (38). The impaired endothelium-dependent relaxation pathway could be related to possible abnormalities in the production and/or activity of endothelium-derived relaxing factors such as NO, PGI₂, and EDHF.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Phenylephrine (Phe)-induced contraction in endothelium-intact (+Endo) and endothelium-denuded (Endo) vascular strips of normal pregnant and reduced uterine perfusion pressure (RUPP) rats.

	NO PRODUCTION DURING PREECLAMPSIA

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

The vascular changes during normal pregnancy have been attributed, in part, to increased NO synthesis by various maternal cells including vascular endothelial cells (7, 17, 32, 44, 111, 112, 151). This is supported by reports that the expression and activity of NO synthase (NOS) is increased in human uterine artery during pregnancy (118). Also, the plasma level, metabolic production, and urinary excretion of cGMP, a second messenger of NO and a cellular mediator of vascular smooth muscle relaxation, are increased during pregnancy (35, 111). Interestingly, cGMP production is markedly increased during the first trimester when the maternal circulation is rapidly vasodilating, whereas the whole body NO production as estimated by the plasma level and urinary excretion of nitrite/nitrate is not proportionately elevated, suggesting additional sources of cGMP (33).

Studies in pregnant experimental animals have also suggested an increase in NO synthesis during late gestation. The endothelium-dependent NO-mediated vascular relaxation is enhanced in late pregnant rats compared with virgin rats (39, 91). Also, the expression of NOS in several tissues, particularly those of the kidney, is elevated during late gestation in rats (1, 5).

The increase in NO production and the reduction of vascular resistance and arterial pressure during normal pregnancy has led investigators to hypothesize that a reduction in NO production could be the cause of the increased vascular resistance and arterial pressure during preeclampsia. In support of this hypothesis, alterations in NO production have been reported in women with preeclampsia (40, 137, 138, 147). Also, NOS blockade with N^G-nitro-L-arginine methyl ester (L-NAME) during mid to late gestation in rats results in pathological changes similar to those observed in women with preeclampsia, such as severe renal vasoconstriction, proteinuria, thrombocytopenia, and intrauterine growth retardation (16, 17, 41, 91, 113, 168). Furthermore, some studies have shown that the arterial pressure is significantly increased in pregnant rats treated with the NOS inhibitor L-NAME compared with virgin rats treated with equal doses of L-NAME (39, 91). However, the question remains whether reduction of NO synthesis is one of the intermediary vascular and cellular mechanisms of the increased vascular resistance and arterial pressure in human preeclampsia and animal models of PIH. If this were the case, one would predict that reduction in uteroplacental perfusion in late pregnant animals, a putative initiating event of PIH (27, 58, 105), would be associated with decreased endothelium-dependent NO-mediated vascular relaxation.

It has been reported that ACh-induced relaxation is reduced in vascular strips of RUPP rats compared with normal pregnant rats (38). Pretreatment of the vascular strips with L-NAME, which blocks NO synthesis, or with methylene blue, which inhibits guanylate cyclase and decreases cGMP production in smooth muscle (83), significantly inhibits ACh-induced vascular relaxation in normal pregnant but not RUPP rats. These studies suggest that NO production or release by endothelial cells and thereby the activity of the NO-cGMP relaxation pathway is reduced in RUPP rats compared with normal pregnant rats (38).

However, whether NO production is reduced in human preeclampsia or in animal models of PIH is not clearly established. Assessment of whole body NO production by measurement of 24 h nitrate/nitrite excretion has yielded variable results. Some clinical studies showed that the nitrite/nitrate levels are reduced in the sera of preeclamptic women (116). Other studies showed that the plasma levels of nitrite/nitrate could be increased during preeclampsia (149). The discrepancy in the nitrite/nitrate levels during preeclampsia could possibly be due to the difficulty in controlling other factors such as nitrate intake. However, in a recent study in preeclamptic women in which dietary intake of nitrate and nitrite was carefully controlled, unequivocal support for reduced NO production could not be demonstrated (33). Also, studies in the RUPP rat model of PIH have shown no significant alterations in total nitrite/nitrate production or urinary excretion (4). These data are difficult to reconcile with the decreased endothelium-dependent vascular relaxation observed in the RUPP rats (38). The apparent dissociation between whole body NO production and the hemodynamic and vascular changes during human preeclampsia and in animal models of PIH can be explained by the possibility that whole body NO production may not be reflective of the relevant vascular NO. Other likely explanations include possible tissue-specific differences in the expression of the NOS isoforms and/or differences in the availability of NO to produce vascular relaxation.

Although the total urinary nitrite/nitrate excretion does not appear to be different between normal pregnant and RUPP rats (4), recent studies suggest that the basal and ACh-induced nitrite/nitrate production in endothelium-intact vascular strips is reduced in RUPP rats compared with normal pregnant rats (15). This may be related, in part, to differential expression of NOS isoforms in various tissues, particularly in the placenta, blood vessels, and the kidney. It has been reported that the expression of NOS is not different in placentas obtained from normal and preeclamptic women (30). However, studies in late pregnant rats showed that the amount of renal endothelial NOS (eNOS) decreases by 39%, whereas the renal inducible NOS (iNOS) and neuronal NOS (nNOS) increase by 31 and 25%, respectively (5). These data raise the interesting possibility that the increased NO production during normal pregnancy in rats is caused by the upregulation of iNOS and nNOS in the kidney and perhaps eNOS in blood vessels. Studies also showed that the expression of the nNOS isoform in renal tissues is reduced in RUPP rats compared with normal pregnant rats (4). Whether the amount of NOS isoforms is altered in blood vessels of RUPP rats compared with normal pregnant rats is unclear and should represent important areas for future investigations.

An emerging area of investigation is whether omitting the vasodilator NO that is derived from any of the NOS isoforms would result in hypertension during pregnancy. Recent studies suggest that NOS gene knockout mice do not become hypertensive during pregnancy (150), perhaps because compensatory vasodilator substances such as prostacyclin may be recruited. However, whether genetic deficiency of any of the NOS isoforms results in PIH in other animal models remains to be investigated.

Also, although the total nitrate/nitrite production may be unchanged in preeclampsia plasma, the availability of NO to produce vascular relaxation may be reduced. Ascorbate is essential for the decomposition of S-nitrothiols and the release of NO. Ascorbate deficiency is typical of preeclampsia plasma and might result in decreased rates of decomposition of S-nitrosothiols. This is supported by reports that preeclampsia plasma contains higher concentrations of total S-nitrosothiols and S-nitrosoalbumin than normal pregnancy plasma. The increase in the total S-nitrosothiol and S-nitrosoalbumin concentrations in preeclampsia plasma may reflect insufficient release of NO from these major reservoirs of NO in this condition (158).

	PROSTACYCLIN PRODUCTION DURING PREECLAMPSIA

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

Although it is recognized that changes in NO production may play a role in some parts of the maternal circulation, there is considerable evidence suggesting additional NO-independent mechanisms (161, 170). Other endothelium-derived relaxing factors such as prostacyclin (PGI₂) may contribute to the hemodynamic and vascular changes observed during normal pregnancy and preeclampsia (Fig. 1). PGI₂ is an anti-platelet aggregator and a vasodilator compound with significant beneficial effects in the maternal circulation during pregnancy. Urinary excretion of 6-keto-prostaglandin F₁ (PGF₁), a hydration product of PGI₂, and 2,3-dinor-6-keto-PGF₁, generated through -oxidation of 6-keto-PGF₁, is increased during normal pregnancy, reaching a maximum during the last month of pregnancy (170).

Alterations in the production of PGI₂ have been reported in women with preeclampsia, thus further suggesting abnormal endothelial cell function during this disorder (10, 62, 164). In women with severe preeclampsia, the excretion of both 6-keto-PGF₁ and 2,3-dinor-6-keto-PGF₁ is lower than in normotensive women during late pregnancy, suggesting that renal PGI₂ synthesis is diminished in preeclampsia (170). Low endothelial generation of PGI₂ has also been suggested in women with preeclampsia (97).

However, the effects of plasma from normal pregnant and preeclamptic women on PGI₂ production by endothelial cells in vitro do not appear to reflect the plasma PGI₂ concentrations in vivo. PGI₂ production by cultured human umbilical vein endothelial cells incubated with plasma from preeclamptic women for 24 h is significantly greater than that by cells exposed to normal pregnancy plasma (45, 46, 51). The differences in endothelial PGI₂ production by plasma from pregnant and preeclamptic women could not be explained by changes in cellular cyclooxygenase and PGI₂ synthase enzyme activity or mass. Instead, the stimulatory effect of preeclampsia plasma on PGI₂ biosynthesis in human umbilical vein endothelial cells appears to be manifested at a step(s) proximal to the activation of cyclooxygenase. Possible mechanisms are increased phospholipase A₂, lipoprotein, or lipid peroxide activities in preeclampsia (51).

The dichotomy between the in vivo reduction in intravascular PGI₂ production that occurs in preeclampsia and the in vitro stimulatory effect of plasma from preeclamptic patients on endothelial cell PGI₂ production could be due to differential effects of acute vs. chronic exposure to the plasma. Recent studies investigated the acute vs. chronic effects of 2% plasma from normal pregnant and preeclamptic women by measuring endothelial PGI₂ production at different time periods of exposure to plasma (11). After 24 h, cells exposed to plasma from preeclamptic women produced more PGI₂ than cells exposed to plasma from normal pregnant women. In contrast, a 72-h exposure to plasma from preeclamptic women resulted in less endothelial cell PGI₂ production than exposure to plasma from normal pregnant women. Thus, in contrast to acute exposure, chronic exposure to plasma from preeclamptic women alters endothelial cells and results in decreased PGI₂ production, an observation consistent with the in vivo findings (11).

Interestingly, in vascular strips of RUPP rats some relaxation to ACh is not completely inhibited by L-NAME or methylene blue (38), suggesting changes in other endothelium-derived vasodilator substances such as PGI₂ in animal models of PIH.

	EDHF PRODUCTION IN ANIMAL MODELS OF PIH

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In addition to enhanced endothelium-dependent NO/PGI₂ synthesis, a hyperpolarizing factor (159) may contribute to the vascular adaptation during normal pregnancy (Fig. 1). Endothelium-derived hyperpolarizing factor (EDHF) has been suggested to play an important role in the enhanced ACh-induced relaxation of small mesenteric arteries of pregnant rats (72). Also, studies on the uterine vascular beds of pregnant rats have suggested that EDHF release is activated by a delayed rectifier type of voltage-sensitive potassium channel (68). Whether EDHF release from vascular endothelial cells is impaired during human preeclampsia or in animal models of PIH remains to be investigated.

	ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN PREECLAMPSIA

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Serum vascular endothelial growth factor (VEGF) immunoreactivity has been shown to be suppressed during normal pregnancy (106). It has also been suggested that serum VEGF may be decreased during preeclampsia (106). However, other studies have shown that the serum VEGF levels are elevated in patients with preeclampsia, suggesting that the growth factor may have a role in the endothelial cell activation/dysfunction that occurs in the disease (13). Maternal plasma VEGF increases before the clinical onset of preeclampsia and is further elevated during the vasoconstricted state observed in this disorder. It has been suggested that the hyperdynamic circulation that characterizes the latent phase of preeclampsia causes vascular shear stress, which in turn increases the levels of circulating VEGF. Because VEGF normally acts as a vasodilator, its increase may represent an unsuccessful vascular rescue response during preeclampsia (19).

	EVIDENCE FOR ENDOTHELIUM-DERIVED CONTRACTING FACTORS DURING PREECLAMPSIA

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

Because an increase in NO production could, in part, explain the reduced vascular reactivity observed during normal pregnancy (17, 32, 44, 112, 151), one would predict that blocking NO synthesis during pregnancy would bring the vascular reactivity back to the level observed in virgin rats. However, the vascular reactivity to Phe in L-NAME-treated pregnant rats is greater than that in virgin rats untreated or treated with L-NAME (39, 91). These data suggest that treatment of pregnant rats with L-NAME not only blocks NO synthesis in endothelial cells, but may also increase the synthesis of vasoactive compounds that would increase vascular reactivity. Thus endothelial cell dysfunction during PIH may be manifested not only as a reduction in vascular relaxation due to decreased endothelium-derived relaxing factors, but also as an increase in vascular reactivity due to increased production of endothelium-derived contracting factors such as endothelin and thromboxane.

	ROLE OF ENDOTHELIN IN PREECLAMPSIA

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

The production of endothelin is increased in women with preeclampsia (25, 52, 123, 157). The concentration of immunoreactive endothelin is elevated in plasma of women with preeclampsia and rapidly returns to a normal pregnancy value within 48 h of delivery, as predicted by the prompt clinical resolution of this disorder. These data suggest that endothelin may contribute to the vasospasm associated with preeclampsia and lend further support to the involvement of endothelial cell dysfunction in the pathophysiology of this disorder (157). Typically, however, the plasma levels of endothelin are highest during the later stage of the disease, suggesting that endothelin may not be involved in the initiation of preeclampsia but rather in the progression of the disease into the malignant hypertensive phase (25, 53, 123, 157).

Experimental studies have also suggested a role for endothelin in mediating the hypertension in animal models of PIH. It has been shown that the increased arterial pressure in animal models of PIH is associated with endothelial cell dysfunction, leading to alterations not only in the synthesis of vasodilators such as NO and PGI₂, but also in the production of endothelin-1 (16, 17, 113, 167). This is supported by reports that long-term inhibition of NO synthesis during late gestation in rats is associated with increased blood pressure and elevated plasma levels of endothelin-1 (59). Also, the expression of preproendothelin is elevated in both the renal cortex and medulla of the RUPP rat model of PIH compared with normal pregnant rats (6). Furthermore, chronic administration of the endothelin A (ET_A) receptor antagonist ABT-627 markedly attenuates the increase in arterial pressure observed in the RUPP rats (6). These studies suggest that endothelin plays a major role in mediating the hypertension produced by chronic reduction of uterine perfusion pressure in pregnant rats.

However, the increased endothelin levels during human preeclampsia and in animal models of PIH may have other vascular effects in addition to promotion of vascular spasm. Endothelin is known to interact with ET_A and ET_B receptors. The interaction of endothelin with specific ET_A and ET_B receptors in smooth muscle initiates a cascade of biochemical events leading to smooth muscle contraction (99, 128, 145, 146, 148, 156). Endothelin also interacts with specific ET_B receptors in the endothelium (128, 145, 146). Basal activation of endothelial ET_B receptors by endothelin and the ensuing release of relaxing factors such as NO, PGI₂, and EDHF have been suggested to promote vascular relaxation and reduce vascular reactivity (73, 142, 145, 146). This is supported by reports that endothelin, via activation of ET_B receptors, could mediate the reduced myogenic reactivity of small renal arteries and the renal vasodilation and hyperfiltration during pregnancy in rats (31, 69). These studies suggest that during preeclampsia an increase in endothelin production and activation of ET_B-mediated vascular relaxation pathways may serve as a rescue mechanism against the excessive increases in vascular resistance and arterial pressure. In relation to these studies, it was shown that a bolus injection of endothelin decreases the arterial pressure in pregnant rats chronically treated with L-NAME. Similar depressor effects are also observed with sarafotoxin S6c, a specific ET_B agonist, and are blocked by the specific ET_B antagonist BQ-788 (109).

	ROLE OF THROMBOXANE IN PREECLAMPSIA

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

Another important endothelium-derived contracting factor is thromboxane A₂ (TXA₂). TXA₂ is released not only from the endothelium, but also from the platelets. TXA₂ is a potent vasoconstrictor with a strong platelet aggregation action. The urinary excretion of TXB₂ metabolites as markers of TXA₂ synthesis is significantly higher in women with preeclampsia than in normotensive pregnant women (63, 64, 164). Also, TXB₂ metabolite excretion correlates with the changes in mean arterial pressure and platelet count, which are indexes of the severity of preeclampsia. Additionally, the excretion of TXB₂ metabolites falls rapidly postpartum parallel with resolution of clinical signs (63). Thus increased TXA₂ biosynthesis appears to correlate with the severity of preeclampsia and may have a pathogenetic role in the disease. These findings have provided a rationale for the use of aspirin in the treatment and prevention of preeclampsia (63). Some clinical studies suggested that low-dose aspirin may attenuate the development of preeclampsia in women at risk for the disease (37). However, other randomized placebo-controlled trials involving women at high risk for preeclampsia showed that low-dose aspirin may have no or only small to moderate benefits when used for prevention of the disease and thus raised questions regarding the validity of this practice (9, 21, 55).

Studies in animal models of PIH also suggested that reduction in placental blood flow during pregnancy and the ensuing endothelial cell dysfunction may increase the production of TXA₂ (167). This is supported by reports that short-term increases in arterial pressure produced by acute reduction in uterine perfusion in pregnant dogs are prevented by TXA₂ receptor antagonists (167).

	ENHANCED VASCULAR SMOOTH MUSCLE REACTIVITY IN ANIMAL MODELS OF PIH

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

Experimental studies have shown that the Phe-induced vascular reactivity in endothelium-intact aortic strips is enhanced in the RUPP rat model of PIH compared with normal pregnant rats. Removal of the endothelium enhances the Phe contraction in pregnant rats, but to a lesser extent in RUPP rats. Also, the Phe contraction in endothelium-denuded vascular strips is still greater in RUPP rats compared with pregnant rats (Fig. 2), suggesting an endothelium-independent component of the increased vascular reactivity in RUPP rats (38).

In addition to the observed pregnancy-associated changes in vascular reactivity in large conduit arteries such as the thoracic aorta (39, 91, 141), recent studies on single smooth muscle cells isolated from resistance renal arteries showed that the Phe-induced cell contraction is reduced in pregnant rats compared with virgin rats but significantly enhanced in pregnant rats treated with L-NAME (115). Although the pregnancy-associated alterations in smooth muscle cell contraction to Phe can be explained by changes in the sensitivity to Phe at the -adrenergic receptor level, they could also be due to changes in the signaling mechanisms downstream from receptor activation.

	CELLULAR MECHANISMS OF VASCULAR SMOOTH MUSCLE CONTRACTION

TOP ABSTRACT INTRODUCTION HEMODYNAMIC AND VASCULAR... PLACENTAL ISCHEMIA AS AN... ENHANCED VASCULAR REACTIVITY IN... ENDOTHELIAL CELL DYSFUNCTION... NO PRODUCTION DURING... PROSTACYCLIN PRODUCTION DURING... EDHF PRODUCTION IN ANIMAL... ROLE OF VASCULAR ENDOTHELIAL... EVIDENCE FOR ENDOTHELIUM-... ROLE OF ENDOTHELIN IN... ROLE OF THROMBOXANE IN... ENHANCED VASCULAR SMOOTH MUSCLE... CELLULAR MECHANISMS OF VASCULAR... VASCULAR SMOOTH MUSCLE [CA2+]I... EVIDENCE FOR ALTERATIONS IN... PKC OF VASCULAR SMOOTH... PHENOTYPIC CHANGES IN VASCULAR... LINKING PLACENTAL ISCHEMIA TO... ROLE OF CYTOKINES AS... ROLE OF OXIDATIVE STRESS... OTHER POSSIBLE PLACENTAL... REFERENCES

It is widely accepted that vascular smooth muscle contraction is triggered by increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to Ca²⁺ release from the intracellular stores and Ca²⁺ entry from the extracellular space (82, 95, 133). Ca²⁺ binds calmodulin to form a complex, which in turn activates myosin light chain (MLC) kinase, causes MLC phosphorylation, initiates actin-myosin interaction and produces smooth muscle contraction (133). However, several laboratories reported dissociations between [Ca²⁺]_i and force (50, 81, 94), between MLC phosphorylation and force, and between [Ca²⁺]_i and MLC phosphorylation and suggested additional regulatory pathways of vascular smooth muscle contraction (132, 155).

In addition to MLC kinase, other protein kinases such as Rho-kinase and mitogen-activated protein kinase have been suggested to contribute to smooth muscle contraction (82, 152). Also, in several cell types, including smooth muscle, the agonist-receptor interaction is coupled to increased breakdown of phosphatidylinositol 4,5-bisphosphate and production of diacylglycerol (DAG), which activates protein kinase C (PKC), an enzyme that enhances the cellular responses to Ca²⁺ (89, 122). Biochemical studies in vascular smooth muscle have shown that PKC is mainly cytosolic under resting conditions and undergoes translocation from the cytosolic to the particulate fraction when the cells are activated by DAG or phorbol esters (89, 122). Also, direct activation of PKC by phorbol esters causes sustained contraction of vascular smooth muscle (42, 96, 131) with no significant change in [Ca²⁺]_i (84, 120). These reports suggested a role for PKC in regulating vascular smooth muscle contraction, at least in part by increasing the Ca²⁺ sensitivity of the contractile proteins. PKC is now known to be a family of Ca²⁺-dependent (, I, II, and ) and Ca²⁺-independent (, , , , , µ, and /) isoforms. These PKC isoforms appear to have different enzyme properties, substrates, and functions and to exhibit different subcellular distributions in the same blood vessel from different species and in different vessels from the same species (85, 87, 93, 104).

	VASCULAR SMOOTH MUSCLE [CA2+]I IN ANIMAL MODELS OF PIH