Molecular and Vascular Targets in the Pathogenesis and Management of the Hypertension Associated with Preeclampsia

Maternal Genes

Female offspring of a pregnancy complicated by PE may have a higher risk of developing PE in their own pregnancies, possibly due to inheritance of PE susceptibility genes [12]. Studies have estimated a 31% heritability for PE and 20% for gestational HTN [13]. A population-based Swedish cohort study has suggested that genetic factors may account for >50% of an individual's susceptibility to PE [14]. Genome-wide linkage studies have identified more than one gene locus on chromosomes 1, 2p13, 2q, 3p, 4q, 9, 10q, 11q23–24, 12q, 15q, 18 and 22q, suggesting that PE is a multigene disorder [15] (Table 1). Also, regions on chromosomes 2p25 and 9p13 may harbor susceptibility genes for PE [16]. A recent study compared the global gene expression in the first trimester placentas from PE and Norm-Preg women and identified 36 major genes in PE placentas, 31 of which were downregulated [17]. Some of the genes implicated in PE include those of VEGF, hypoxia-inducible transcription factor (HIF), Fas, and leptin.

Subjects carrying the T allele VEGF 936C/T genotype have lower VEGF plasma levels than subjects carrying the VEGF 936C/C genotype, and changes in VEGF gene and plasma levels could play a role in the vascular endotheliosis observed in PE [18].

HIF-1α and -2α proteins and regulated genes are increased in PE placenta. Upregulation of placental HIF-1α may induce the expression of the various factors that are secreted into the maternal circulation e.g. soluble fms-like tyrosine kinase 1 (sFLT-1) and soluble endoglin (sEng), which lead to disruption of the endothelium and other manifestations of PE. Accumulation of HIF-1α proteins in PE placenta may be due to both increased formation secondary to ischemia/hypoxia and reduced degradation after reperfusion/oxygenation as a result of proteasomal dysfunction [19].

The Fas ligand-Fas is a known pathway of apoptosis. The binding of Fas ligand to Fas triggers the activation of caspases 3, 7 and 9 and leads to apoptotic event. Polymorphisms of Fas genes and Fas ligand genes may play a role in PE. In Norm-Preg, activated T lymphocytes, which recognize paternal antigens, express Fas and interact with the cytotrophoblasts expressing the Fas ligand, leading to apoptosis of T cells and reduction in their ability to recognize and destroy the cytotrophoblasts invading the myometrium and spiral arteries. In PE, the expression of Fas 670 G variant is associated with reduced Fas production in activated T lymphocytes, leading to reduced T cell apoptosis, increased destruction of cytotrophoblasts, and inadequate invasion of spiral arteries [20].

An increase in circulating levels of leptin and leptin gene polymorphism may be associated with PE [21]. Recent data have demonstrated increased leptin expression in the basal plate of PE placenta compared with Norm-Preg control tissue. Also, genes encoding sialic acid binding Ig-like lectin 6 (Siglec-6), a potential leptin receptor, and pappalysin-2 (PAPP-A2), a protease that cleaves insulin-like growth factor (IGF) binding proteins, are over-expressed in PE [2], supporting a link between increased leptin expression and PE.

Fetal Genes

Another factor in the development of PE is fetal genes. PE is more common in women homozygous for the inhibitory killer immunoglobulin-like receptor (KIR) haplotypes (AA) than in women homozygous for the stimulator KIR halpotypes (BB), and the effect is strongest if the fetus is homozygous for the human leukocyte antigen class II (HLA-C2) haplotype [29]. Also, gene-to-gene interaction between fetal HLA-G and maternal KIR2DL4 is associated with PE risk in multigravid pregnancy [30]. In mice, mutations in the cyclin-dependent kinase inhibitor cdkn1c (p57^Kip2), a regulator of embryonic growth, induces changes in the placenta architecture indicative of the RUPP found in HTN-Preg. Also, pregnant mice expressing wild-type levels of p57^Kip2 develop a PE-like condition when carrying p57^kip2-deficient pups, supporting a role for fetal genes in the development of PE [15].

Paternal Genes

Paternal genes could play a role in the development of PE. Studies compared men whose mothers had PE to men whose mothers did not have PE and found that a paternal family history of PE was associated with more than 2-fold increase in risk of PE [31,32]. PE is also common in hydatid-form mole-pregnancies in which all the fetal chromosomes originate from the father [33].

Ethnic Background and PE

Maternal ethnicity may be a factor in the development of PE. African-American women have the highest incidence rate of PE (5.2%), while Asian women have the lowest rate (3.5%) [34]. Paternal ethnicity may also play a role in the incidence of PE. Among African-American women the incidence of PE is greater if paternal ethnicity is different from maternal ethnicity (5.8%) than if the paternal and maternal ethnicities are the same (5.2%). Among Asian women the incidence of PE is greater if the paternal ethnicity is different (4.6%) than if it is the same as maternal ethnicity (3.2%). In contrast, among Native-American women the incidence of PE is greater if the paternal ethnicity is the same (9.7%) than if it is different from maternal ethnicity (3.3%) [35].

Calcium, Magnesium, Potassium

Ca²⁺ concentration in the extracellular fluid is strictly maintained at a normal serum level of 2.2–2.6 mmol/L. 40% of serum calcium is bound to albumin, 10% in complex with citrate and the rest is ionized Ca²⁺ – the most important fraction [36]. During pregnancy, Ca²⁺ is transferred to the fetus through the placenta. The higher Ca²⁺ requirement during pregnancy requires physiologic adaptation of the maternal Ca²⁺ homeostatic mechanisms including intestinal absorption, urinary excretion, and Ca²⁺ turnover in the bones. Ca²⁺ deficiency may increase the risk of PE particularly among teenagers, and Ca²⁺-supplementation may reduce the incidence of PE in this age group. The beneficial effects of Ca²⁺-supplementation may depend not only on the mother’s age but also on the socioeconomic status, particularly in geographical locations where Ca²⁺ intake is low [36]. Some studies have suggested that Ca²⁺- supplementation in the range of 375–2000mg/day in a population with low Ca²⁺ intake is a safe, effective and inexpensive measure to reduce the risk of HTN-Preg and PE [37]. However, in one study, although women who ingested Ca²⁺ tablets 2000 mg/day had a 66.7% lower risk of PE, among women who developed PE there was no significant reduction in the severity of the disease [38]. Also, the Ca²⁺ for PE Prevention (CPEP) clinical trial has shown no effect of 2000 mg/day Ca²⁺-supplementation on BP or incidence of PE [39]. Experimental studies suggest beneficial effects of dietary Ca²⁺ during pregnancy. In pregnant ewes, restricted Ca²⁺ intake is associated with decreased plasma Ca²⁺ levels and uterine blood flow, and increased BP and urinary protein; all symptoms similar to those of PE [40]. Also, low Ca²⁺ intake in pregnant rats is associated with increased pressor effects of AngII [41]. In both Norm-Preg and nonpregnant rats, a high Ca²⁺ diet (1.7%–2.1%) is associated with reduction in BP, and attenuated VSM reactivity [42]. Thus, Ca²⁺-supplementation may have the strongest influence on PE when dietary Ca²⁺ is low. If Ca²⁺ intake is adequate, no Ca²⁺-supplementation may be needed [36].

Magnesium (Mg²⁺) is an essential dietary mineral and a cofactor for many enzymes that regulate temperature and protein synthesis. Mg²⁺ also maintains the membrane potential in nerves and muscle [43]. Observational studies of medical records have suggested that Mg²⁺-supplementation during pregnancy reduces the risk of IUGR and PE. Maternal Mg²⁺ intake is also positively associated with birth weight. Diets high in potassium and fiber are also associated with reduced risk of HTN [44]. Thus, maternal intake of recommended amounts of Ca²⁺, Mg²⁺, potassium and fiber may reduce the risk of PE.

Vitamins

Vitamin D (cholecalciferol) is a major cofactor in Ca²⁺ absorption and metabolism. Cholecalciferol is formed in the skin during exposure to sunlight and UV irradiation and absorbed from ingested food, then converted in the liver to 25(OH) cholecalciferol or 25(OH)D, and further hydroxylated by 1α-hydoxylase in the kidney to 1,25 dihydroxycholecalciferol or 1,25(OH)₂D3, the most active form of vitamin D. Adequate levels of 25(OH)D (>80 nmol/L) are needed for optimal health of the skeletal system, and may influence the cardiovascular system and BP. 1,25(OH)₂D3 levels are elevated during pregnancy due to increased activity of renal 1α-hydoxylase and placental production. Also, vitamin D receptors are found in the placenta [36]. Vitamin D deficiency and altered Ca²⁺ metabolism have been implicated in abnormal placentation and PE [45]. Circulating 1,25-(OH)₂D3 levels in both maternal and umbilical cord compartments are lower in PE compared with Norm-Preg [46]. Vitamin D receptor null mice show an increase in renin expression, increased plasma AngII, and HTN. Also, in wild-type mice, inhibition of 1,25(OH)₂D3 synthesis leads to increased renin expression, and 1,25(OH)₂D3 injection causes renin suppression, supporting a role of 1,25(OH)₂D3 in the regulation of the renin-angiotensin system (RAS) and BP [47].

Vitamins may influence the oxidants and antioxidants balance and the risk of PE. Serum levels of vitamins E and C are decreased in women with PE, and vitamin supplementation may decrease the incidence of PE in women at high risk [48]. Folic acid or folate is a coenzyme in the production of nucleic acids and cell growth. Folic acid may play a role in the implantation and development of placenta and could reduce the risk of PE by improving EC function in the placenta and systemic vessels and by lowering plasma homocysteine level [49]. In a recent study, women who received multivitamins containing folic acid demonstrated increased serum folate, lower plasma homocysteine, and reduced risk of PE by 63% compared to women with no supplementation [50].

Obesity

The prevalence of obesity is rapidly increasing worldwide. In the United States, 28% of women aged 20–39 years are obese and 54% are either obese or overweight [51]. The risk of PE rises strikingly with the increase in pre-pregnancy body mass index (BMI). Compared with women with a BMI of 21, the risk of PE doubles at a BMI of 26, triples at a BMI of 30 and increases further with severe obesity [52]. In a study assessing the risk factors of severe PE, other than a history of PE, severe obesity (BMI >32.2) was the major maternal risk factor [53]. Obesity is associated with low-grade inflammation and increased circulating inflammatory markers [54]. Plasma levels of C-reactive protein, TNF-α, IL-6 and IL-8 are elevated in obese subjects, and body fat is a possible source of these inflammatory markers. Markers of inflammation are also increased in the vasculature of obese women and this may contribute to Vascular Targets in the vascular changes associated with PE. Obesity is also associated with increased circulating levels of leptin, and a leptin gene polymorphism has been linked to increased risk of developing PE [21].

Adiponectin, a hormone produced abundantly by adipose tissue, plays a role in obesity-related inflammation. In women with normal body weight (BMI <25 kg/m²), an increase in adiponectin levels may suppress the expression of adhesion molecules in vascular ECs and cytokine production from macrophages in order to minimize the inflammatory process associated with PE. However, in obese women (BMI ≥25 kg/m²) with PE, this inflammation-minimizing mechanism may fail and the adiponectin concentration is reduced [55].

Ethnic differences in obesity may influence the incidence of PE. African-American women are more likely to be overweight prior to pregnancy and to gain the required weight during pregnancy, while Asian women gain less than the required weight [56]. Thus, ethnicities most likely to gain weight during pregnancy correlate with those most at risk of developing PE.

Diabetes

During Norm-Preg, women experience brief insulin resistance and glycemia after a meal. Increased sugar consumption in pregnant women may lead to hyperglycemia, which in turn inhibits endothelium-dependent vasorelaxation. The risk of PE is increased in women with family history of type 2 diabetes or pre-pregnancy hyperinsulinemia and insulin resistance [57]. Also, the prevalence of metabolic syndrome (characterized by visceral obesity, insulin resistance, hyperglycemia, type 2 diabetes, dyslipidemia, and high BP) is 3-fold higher in PE women than in women with gestational diabetes [58]. Women with metabolic syndrome have endothelial dysfunction likely because their blood vessels are more sensitive to the circulating factors released during PE. The rate of PE is greater in women with pre-pregnancy diabetes (~12%) than in women without diabetes (~3%). Also, the incidence rate of PE with abruption or infarction of placenta in women with pre-pregnancy diabetes (2.2%) is greater than that in women without diabetes (1.8%) [59]. Gestational diabetes is also associated with high BMI and increased risk of PE [60].

Vascular Endothelial Growth Factor (VEGF)

VEGF is a major angiogenic factor and a prime regulator of EC proliferation, vasculogenesis and vascular permeability [103]. VEGF may also have vasodilator effects by stimulating the NO-cGMP vascular relaxation pathway. VEGF increases EC intracellular free calcium concentration ([Ca²⁺]_i), which promotes calmodulin binding to eNOS and thereby stimulates NO production [104]. The gene encoding VEGF is located on chromosome 6 band p21 and comprises a 14 kb coding region with 8 exons and 7 introns [105]. VEGF binds to Flt-1 (VEGFR1) and flk1 (KDR VEGFR2), both are tyrosine kinase receptors present on EC membrane [103]. VEGF (or VEGF-A) belongs to a gene family that includes placental growth factor (PLGF), VEGF-B, VEGF-C and VEGF-D. While the circulating total VEGF levels are elevated in PE, the free (unbound) VEGF concentration is reduced possibly due to the elevated levels of sFlt1 receptor, which captures free VEGF [95,106]. Patients receiving VEGF antagonists such as bevacizumab for renal cell carcinoma develop HTN, proteinuria and endothelial activation resembling PE [107]. Also, mice lacking one VEGF allele in renal podocytes develop the typical renal pathology found in PE [107]. In non-pregnant mice, infusion of antibodies against VEGF to cause a 50% reduction of VEGF leads to glomerular endotheliosis and proteinuria similar to what is seen in PE [107,108]. Also, in vitro angiogenesis studies have shown that exogenous VEGF or an antibody against sFlt1 can reverse the anti-angiogenic effects of PE plasma [109]. These data support the contention that the VEGF levels and signaling pathways may be altered during PE.

Soluble fms-like tyrosine Kinase-1 (sFlt-1)

sFlt-1 is a VEGF and PlGF antagonist protein that binds VEGF and prevents its interaction with its endogenous receptors. sFlt1, which lacks the transmembrane and cytoplasmic domains, is made in large amounts by the PE placenta and is released into the maternal circulation [110]. Circulating sFlt1 levels are increased in women with established PE and may begin to rise before the onset of clinical symptoms [76]. Consistent with the antagonistic effect of sFlt1, free (or unbound) VEGF and PlGF concentrations are decreased in PE women at disease presentation and even before the onset of clinical symptoms [76,95]. Increases in maternal sFlt1 are observed in several conditions that are considered risk factors for PE. For example, in twin pregnancy, which is associated with a 2 to 3-fold increase in the incidence of PE [111], circulating sFlt1 levels and sFlt1/PlGF ratios are twice as high as those in singleton pregnancy [112]. Also, the Flt-1/sFlt1 gene is located on chromosome 13q12, and in trisomy 13 an extra copy of this gene may lead to excess circulating sFlt1, reduced free PlGF level, and increased sFlt1/PlGF ratio and thereby contribute to the increased risk of PE [113]. In support of a role of sFlt-1 in PE, exogenous sFlt-1 given to pregnant rats results in HTN, proteinuria and glomerular endotheliosis, akin to the PE phenotype observed in human [95]. Also, in tissue culture, exposure of endothelial cells to conditioned medium containing PE plasma results in reduced angiogenesis, while removal of sFlt-1 by immunoprecipitation and adding the supernatant restores EC function and angiogenesis to normal levels [109].

Endoglin

Endoglin (Eng) or CD105, a co-receptor for TGF-β1 and TGF-β3, is highly expressed on cell membranes of vascular ECs and syncytiotrophoblasts [114]. Placental endoglin is upregulated in PE, releasing soluble endoglin (sEng) into the maternal circulation [115]. sEng is an anti-angiogenic protein that inhibits TGF-β1 signaling in the vasculature [115]. Circulating sEng levels increase beginning 2 to 3 months before the onset of PE and more steeply in women who develop PE, and peak at the onset of clinical disease [76]. sEng may act in concert with sFlt-1 to cause EC dysfunction, resulting in severe PE and/or HELLP syndrome.

sEng disrupts formation of endothelial tubes in human umbilical vein endothelial cells (HUVECs) in culture and induces vascular permeability and HTN in pregnant rat in vivo [115]. Mutations in endoglin result in loss of capillaries, arteriovenous malformations, and hereditary hemorrhagic telangiectasia [6]. Pregnant rats with overexpression of both sEng and sFlt-1 demonstrate proteinuria, severe HTN, HELLP syndrome, and IUGR. Histological analysis of tissues from these pregnant rats reveals severe glomerular endotheliosis, infarction in the placenta, necrosis in the liver, and fragmented red blood cells (schistocytes) [115]. Also, placental ischemia in the RUPP rat model is associated with increased expression of sEng and HIF-α and shifts the balance of angiogenic factors in the maternal circulation toward an angiostatic state [77].

Placental Growth Factor (PlGF)

PlGF is a pro-angiogenic factor of the VEGF family that may play a role in EC growth and the control of vasculogenesis, angiogenesis, and placental development during pregnancy [85,95,109]. During Norm-Preg, circulating PlGF levels are more than 40 times greater than VEGF, but PlGF has only 1/10^th the affinity for the Flt-1 receptor compared to VEGF. During PE, circulating PlGF levels decrease while the levels of its antagonist protein sFlt-1 increase [116]. The sFlt-1/PlGF concentration ratio is much higher in PE than Norm-Preg women as early as the second trimester, and therefore can be used as an early indicator of the onset of PE [117]. However, the use of sFlt-1 and sEng as mediators or predictors of PE should be viewed with extreme caution as the levels of sFlt-1 and sEng may not be altered in some women or experimental animals with PE/HTN-Preg as compared to Norm-Preg.

Cytokines

During Norm-Preg, women may have elevated inflammatory response and increased plasma levels of plasminogen activator inhibitor-1 (PAl-1), TNF-α and C-reactive protein. In PE, the ischemic placenta may contribute to the maternal EC dysfunction by enhancing the synthesis of TNF-α, IL-6, and IL-8. TNF-α enhances the release of ET-1 from ECs and reduces acetylcholine-induced vasodilatation [118]. TNF-α induces oxidative damage as it destabilizes electron flow in mitochondria, resulting in the release of oxidizing free radicals and formation of lipid peroxides that damage ECs [118]. The plasma level of TNF-α and IL-6 increases 2- to 3-fold in women with PE compared with third-trimester Norm-Preg and women with gestational HTN [118]. In contrast, IL-10, which may be involved in the maintenance of pregnancy by inducing corpus luteum maturation and progesterone production, is decreased in PE women [119]. RUPP during pregnancy and the ensuing placental ischemia are thought to increase the release of TNF-α and IL-6 into the maternal circulation, leading to generalized EC dysfunction and HTN. Also, chronic infusion of TNF-α or IL-6 in late pregnant rats causes significant elevation of BP, enhanced vascular contraction and reduced endothelium-dependent vascular relaxation possibly due to inhibition of the endothelial NO-cGMP pathway [80,120].

Reactive Oxygen Species (ROS)

Pregnancy is a state of oxidative stress characterized by placental production of ROS such as superoxide (O₂^•–) and hydrogen peroxide (H₂O₂). In Norm-Preg, the production of ROS is counterbalanced by abundant antioxidant defenses. However, in PE excessive ROS production overpowers antioxidant defenses, leading to increased oxidative stress and ROS including hydroxyl radicals (–OH) and peroxynitrite (ONOO–) [121]. Increased levels of thromboxane and lipid peroxides and loss of glutathione peroxidase (GPx) activity have been demonstrated in placentas from PE compared with Norm-Preg women [122]. Also, mRNA levels of ROS modulators such as hemoxygenese (HO)-1, (HO)-2, copper/zinc superoxide dismutase (SOD), GPx and catalase are decreased in blood cells of PE compared to Norm-Preg women and the reduction in the amount of HO-1 and HO-2 correlates with the severity of PE [123].

Neutrophils represent an important source of ROS and EC damage in PE. Stimulated neutrophils from women with PE produce more O₂^•– compared to those from Norm-Preg women. Neutrophils also produce NO, which can protect cells from O₂^•–-induced damage in Norm-Preg. However, in PE, O₂^•– production may dominate the neutrophil and EC NO production, resulting in EC injury [88].

ROS may be involved in the severe endoplasmic reticulum stress initiated by deficient remodeling of the uterine arteries during PE [124]. The decreased formation of spiral arteries causes them to spontaneously constrict, resulting in ischemia-reperfusion injury and increased in ROS, which decreases the amount of stored Ca²⁺ and intracellular ATP and thereby produces misfolded proteins. These misfolded proteins create endoplasmic reticulum stress, which the cell attempts to fix via the unfolded protein response (UPR). During low-grade endoplasmic reticulum stress, the UPR promotes phosphorylation of eukaryotic initiation factor 2, which prevents the binding of the initiator to the ribosome and reduces cell proliferation, and thereby maintains cell survival. However, if the endoplasmic reticulum stress is severe enough, the prolonged exposure to UPR during pregnancy makes the cells undergo apoptosis. As a result, syncytiotrophoblast microvillous membranes (STBM) particles enter the maternal circulation, impair vascular EC function, and lead to PE and IUGR [124].

O₂^•– and peroxynitrite (ONOO^–) may promote the vascular dysfunction in PE through stimulation of MMP-1, -2, and -9 [125] and degradation of HIF-1α in VSM [126]. ROS increase VSM [Ca²⁺]_i by stimulating the influx of extracellular Ca²⁺, inhibiting Ca²⁺-ATPase activity, and promoting inositol trisphosphate (IP₃)-induced Ca²⁺ release from the intracellular stores. ROS also activate protein kinase C (PKC) and promote vasoconstriction by activating Rho-A/Rho-kinase signaling mechanisms [127].

Hypoxia Inducible Factors (HIF)

HIF-1 is a transcriptional activator that plays a role in the physiologic responses to hypoxia, and the pathophysiology of ischemic cardiovascular disease, PE and IUGR. HIF-1 is a heterodimer consisting of an oxygen-regulated HIF1-α and HIF2-α subunits and a constitutively expressed HIF1-β subunit [128]. To date, more than 100 HIF-1 downstream genes with varying functions have been identified including VEGF, leptin, TGF-β3, and NOS. Also, DNA microarrays have shown that more than 2% of all human genes are regulated by HIF-1 in arterial ECs, directly or indirectly [129]. HIF-1 activates the expression of these genes by binding to a 50-base pair cis-acting hypoxia response element located in their enhancer and promoter regions. Under normoxic conditions, the α-subunit of HIF-1 is rapidly degraded by ubiquitination and proteosomal degradation [130]. Because placental hypoxia may play a role in PE, the role of HIFs has been investigated. The expression of HIF-1α and HIF-2α, but not HIF-1β, is selectively increased in PE placenta. Accumulation of HIF-1α protein occurs as a result of both increased formation secondary to relative ischemia/hypoxia and reduced degradation after reperfusion/oxygenation due to proteosomal dysfunction [19]. The molecular mechanisms underlying the changes in HIF expression and the genes affected during PE are unclear. However, upregulation of HIF-1 has been shown to enhance the expression of VEGF [126]. Also, a HIF-1α binding site exists in the promoter region of the ET-1 gene and may act to regulate ET-1 expression and consequently vasoconstriction [131].

2-Methoxy estradiol (2-ME) Deficiency

2-ME, a natural metabolite of E2, is elevated during the third trimester of Norm-Preg. Plasma levels of 2-ME are markedly lower in women with severe PE. Pregnant mice deficient in catechol-O-methyltransferase (COMT) demonstrate a PE-like phenotype resulting from the absence of 2-ME. 2-ME ameliorates PE-like features in the COMT-deficient pregnant mice and suppresses placental hypoxia, HIF-1α expression and sFLT-1 elevation. Measurement of 2-ME in plasma and urine may be used as a diagnostic marker for PE, and 2-ME may prevent or treat PE [83].

Nitric Oxide (NO)

NO is a potent vasodilator and relaxant of VSM. NO is produced from the transformation of L-arginine to L-citrulline by neuronal nNOS, inducible iNOS, and endothelial eNOS. NOS expression and activity are increased in human uterine artery and NO production is enhanced during Norm-Preg [135,136]. Also, the plasma concentration and urinary excretion of cGMP, a second messenger of NO and cellular mediator of VSM relaxation, are increased in Norm-Preg [137]. Because NO is an important vasodilator in Norm-Preg, NO deficiency during PE might be involved in the disease process.

Measurements of total NO production during HTN-Preg have not produced consistent findings [137]. Some studies have shown that the concentration of nitrate/nitrite, the stable end product of NO, is reduced in the sera of women with PE [138], while other studies have shown increased levels [139]. The difference in the nitrate/nitrite levels during PE could be related to dietary nitrate intake. While measurements of total nitrate/nitrite did not produce consistent findings, vascular endothelial NO production may be reduced during PE [140]. This is supported by report that NOS blockade with N^ω-nitro-L-arginine methyl ester (L-NAME) during mid- to late gestation in rats produces pathological changes similar to those observed in women with PE including increased BP, renal vasoconstriction, proteinuria, thrombocytopenia and IUGR [141]. Also, chronic RUPP in pregnant rats causes significant increase in BP, proteinuria, decreased GFR and renal plasma flow (RPF), and IUGR. Although the RUPP rat does not demonstrate significant reduction in whole-body NO synthesis as assessed by urinary excretion of nitrate/nitrite, significant reduction in the renal expression of nNOS may contribute to the changes in renal hemodynamics and the HTN in this experimental model of PE [98].

Prostacyclin (PGI₂)

PGI₂ is an anti-platelet aggregator and a vasodilator produced from the metabolism of arachidonic acid by the cyclooxygenase (COX)-1 and COX-2. Endothelium-derived PGI₂ may contribute to the hemodynamic and vascular changes during pregnancy. During Norm-Preg, the plasma and urinary levels of 6-keto-prostaglandin F1α (PGF1α), a hydration product of PGI₂, are increased [142]. Hypoxia causes downregulation of COX-1 and may alter PGI₂ production in PE [143]. Plasma and urinary concentrations of PGF1α are lower during severe PE than in Norm-Preg, suggesting that overall PGI₂ synthesis is diminished. Endothelial PGI₂ production may also decrease in PE [144]. A recent study has shown that the release of PGI₂ is not different in apical and basal trophoblasts of PE compared with Norm-Preg women. On the other hand, the release of thromboxane A₂ (TXA₂) from basal trophoblast cells was increased in PE women, suggesting that it may contribute to the increased placental vasoconstriction in PE [145].

Endothelium-Derived Hyperpolarizing Factor (EDHF)

EDHF is an important relaxing factor particularly in the small resistance vessels. Also, while NO and PGI₂ are the dominant vasodilators in male mice, EDHF may be the predominant relaxing factor in female mice [146]. Studies in subcutaneous arteries from PE women demonstrated that vasodilatation of EDHF are mediated either by myoendothelial gap junctions alone or in combination with H₂O₂ or CYP450 epoxygenase metabolites of arachidonic acid [147]. Other studies on the uterine vessels of pregnant rats have suggested that EDHF release is activated by a delayed rectifier type of voltage-sensitive K⁺ channel [148]. Although NO, PGI₂ and EDHF have different vasodilator mechanisms, they may affect each other. For example, PGI₂ may upregulate eNOS and NO production in ECs, and activate the inward rectifier K⁺ channels through the release of EDHF [149,150].

Endothelin-1 (ET-1)

ET-1 is produced by ECs and acts on VSM to cause vasoconstriction. Hypoxia induces the synthesis and secretion of ET-1 from ECs [131]. The plasma concentration of ET-1 could reach ≈2- to 3-fold higher in PE than Norm-Preg women [140]. Also, ET-1 secretion is 4- to 8-fold higher in umbilical cord ECs of PE than Norm-Preg women [151]. The elevated plasma levels of ET-1 in PE women return to normal levels within 48 h of delivery, suggesting that ET-1 may contribute to the vasoconstriction and HTN in PE [152]. Typically, plasma levels of ET-1 are highest during the later stage of the disease, suggesting that ET-1 may not be involved in the initiation of PE, but rather in the progression of the disease into a malignant phase [93].

Although the circulating levels of ET-1 may not dramatically increase during PE, the role of ET-1 as a paracrine or autocrine agent in the vasculature should be considered. ET-1 interacts with ETAR and ETB₂R in VSM and leads to vascular contraction. ET-1 stimulates Ca²⁺ release from the intracellular stores, Ca²⁺ influx through voltage-dependent Ca²⁺ channels, and PKC-mediated Ca²⁺-sensitization pathways of VSM contraction [153]. ET-1 also activates ETB₁R in ECs and causes the release of vasodilator substances such as NO, PGI₂ and EDHF [154]. ET-1, via activation of ETB₁R, may mediate the reduced myogenic reactivity and vasodilation of renal arteries as well as hyperfiltration during pregnancy in rats [155]. Downregulation of ETBR may predispose women to PE by impairing trophoblast invasion. Also, excessive expression of ET-1 or ETAR or ETB₂R on VSM of renal arterioles may overwhelm the vasodilatory pathway initiated by the pregnancy hormone relaxin [156]. Studies have shown that increasing AT1-AA in pregnant rats to the levels observed in PE women increases BP, and that the HTN is attenuated by oral administration of the AT₁R antagonist losartan or an ETAR antagonist, supporting a role of ET-1 in PE [101].

Angiotensin II (AngII)

AngII is an important circulating hormone for BP regulation and electrolyte homeostasis. Also, ~40% of AngII is produced locally in the placenta by chymase, a chymotrypsin-like serine protease, which is non-angiotensin converting enzyme and produced mainly from villous syncytiotrophoblast [157]. AngII, via endothelial AT₂R, stimulates the release of NO through activation of eNOS [158] as well as the synthesis of PGI₂ [159], which oppose AngII-induced vasoconstriction. AngII, via AT₁R, causes vascular remodeling by promoting vasoconstriction, vascular growth, and inflammation. AngII increases VSM [Ca²⁺]_i and activates Rho/Rho-kinase, a prominent regulator of VSM contraction, the cytoskeleton and vascular remodeling [160]. TNF-α stimulates AngII production in the female reproductive tract [161] and IL-6 upregulates the expression of AT₁R in VSM cells [162].

Norm-Preg is associated with increased plasma renin and AngII levels, but decreased sensitivity and blunted response to AngII. The reduction in AngII sensitivity during Norm-Preg could be related to the relative expression of AT₁R and AT₂R, and the absence of adaptive changes in the AngII receptor expression could contribute to PE [163]. During Norm-Preg monomeric AT₁R are inactivated by ROS leading to lower AngII sensitivity. In PE, AT₁R forms a heterodimer with the bradykinin B2 receptor and thereby shows resistance to ROS inactivation and remains active and hyper-responsive to AngII [164]. AngII peptide levels, renin, and angiotensin converting enzyme (ACE) mRNA are also higher in the uterine placental bed in PE compared with Norm-Preg controls [165].

Agonistic autoantibodies to AT₁R (AT1-AA) may provide a link between placental ischemia and HTN-Preg [166]. AT1-AA induces signaling in vascular cells and activates protein-1, calcineurin, and nuclear factor kappa B (NFκB) [166]. Also, activation of AT₁R by AT1-AA in human trophoblasts may increase PAI-1 production and cause shallow trophoblast invasion [167]. Administration of AT1-AA to pregnant rats increases ET-1 levels in renal cortex and placenta [101]. Also, AT1-AA can additively contribute, alongside AngII and local placental hypoxia, to the excess sFlt-1 secretion in PE [166].

Plasma hemopexin activity increases during Norm-Preg from 10 weeks onward and active hemopexin downregulates the AT₁R in human monocytes and ECs in vitro, and in aortic rings of Norm-Preg rats. It has been suggested that inhibition of hemopexin activity during PE may enhance AT₁R expression and promote vascoconstriction [168].

Thromboxane A₂ (TXA₂)

TXA₂ is a potent stimulator of platelet aggregation, vasoconstriction, and VSM cell proliferation and mitogenesis [99,169]. PE is associated with deficient vascular production of PGI₂ and excessive production of TXA₂, and this imbalance might explain some of the manifestations of PE such as HTN and platelet aggregation. Measurements of urinary metabolites of TXB₂, markers of TXA₂ synthesis, and PGI₂ have shown an imbalance in their levels that predates the clinical symptoms of PE [122]. These findings led to the suggestion that antiplatelet agents such as low-dose aspirin and thromboxane modulators might be effective in preventing PE [170]. However, clinical trials on women at high risk for PE showed no benefit of low dose aspirin when used as a preventive measure [171]. Another study has shown that ozagrel, a thromboxane modulator, reduces the occurrence of PE, HTN-Preg and proteinuria [172], making it important to further examine the role of TXA₂ in PE.

VSM Ca²⁺

Ca²⁺ is a major determinant of VSM contraction and growth. VSM contraction is triggered by increases in [Ca²⁺]_i due to Ca²⁺ release from the intracellular stores and Ca²⁺ entry from the extracellular space. Ca²⁺ binds calmodulin to form a complex which activates myosin light chain (MLC) kinase, causes MLC phosphorylation, initiates actin-myosin interaction and produces VSM contraction [173]. Endothelium-derived relaxing factors act on VSM to inhibit phospholipase C, open K⁺ channels or stimulate Ca²⁺ extrusion, and thereby decrease [Ca²⁺]_i. EC dysfunction is associated with decreased release of relaxing factors, and decreased VSM Ca²⁺ extrusion mechanisms. EC dysfunction also causes an increase in VSM contracting factors, which stimulate Ca²⁺ mobilization. Studies in myometrial and subcutaneous resistance microvessels demonstrated that the reactivity to high KCI depolarizing solution, phenylephrine (PHE) and AngII is not increased in PE compared with Norm-Preg women, and suggested that this is an unlikely mechanism of the increased vascular resistance in PE [174]. On the other hand, the basal and agonist-stimulated [Ca²⁺]_i are reduced in renal arterial VSM cells from Norm-Preg compared with virgin rats, but significantly elevated in RUPP rats and pregnant rats treated with L-NAME [81,102]. AngII- and caffeine-induced transient contraction and [Ca²⁺]_i in Ca²⁺-free solution are not different in VSM cells from Norm-Preg and RUPP rats, suggesting that the pregnancy associated changes in VSM contraction may not involve the Ca²⁺ release from the intracellular stores. In contrast, the AngII- and KCl-induced maintained [Ca²⁺]_i in Ca²⁺-containing medium is greater in VSM of RUPP than Norm-Preg rats, suggesting that the Ca²⁺ entry mechanisms of VSM contraction are enhanced in animal models of HTN-Preg [102].

VSM Protein Kinase C (PKC)

Activation of PKC by phorbol esters causes sustained contraction of VSM with no change in [Ca²⁺]_i, suggesting that PKC increases the Ca²⁺ sensitivity of the contractile proteins. Vascular PKC activity does not change during early and mid-gestation, but is reduced in late pregnant compared with virgin rats, ewes and gilts [175,176]. The changes in vascular PKC activity during late pregnancy could be related to the increased NO and cGMP production. Vascular PKC activity is greater in L-NAME treated pregnant rats than virgin rats, suggesting that L-NAME treatment not only inhibits NO synthesis, but may also increase the synthesis of vasoactive compounds that increase PKC activity [175]. Increased VSM PKC expression/activity has been identified in HTN [177]. Also, the Ca²⁺ sensitivity of VSM contractile myofilaments is increased in women with PE, and PKC activation may be involved in the vascular changes observed in PE [178]. PKC may also play a role in the changes in AngII and AT₁R-mediated signaling associated with PE. Studies on cultured neonatal rat cardiomyocytes have shown that IgG in plasma from PE women enhances AT₁R-mediated chronotropic response, and treatment of cardiomyocytes with the PKC inhibitor calphostin C prevented the stimulatory effect of PE IgG. Also, examination of VSM cells with confocal microscopy have shown colocalization of purified IgG from PE women and AT1-AA, and suggested that PE women may develop stimulatory AT1-AA via a PKC-mediated process [179]. The expression and activity ofα- and δ-PKC are also enhanced in L-NAME treated pregnant compared with Norm-Preg rats, supporting a role of PKC in the increased vasoconstriction and vascular resistance observed in PE [175].

VSM Rho/Rho-Kinase

Rho is a family of small GTP-binding proteins that may regulate cell proliferation, migration, cytoskeletal reorganization and contraction. During cell activation by growth factors and vasoactive substances Rho-kinase is activated by GTP binding and inactivated by hydrolyzing GTP to GDP [180]. Rho-kinase may play a role in the increased Ca²⁺ sensitivity of the contractile proteins observed in subcutaneous resistance arteries from PE compared with Norm-Preg and nonpregnant women [178]. Also, stimulation of AT₁R induces upregulation of RhoA/Rhokinase activity in hypertensive rats, and an increase in AngII activity during PE may activate Rho-kinase and promote vasoconstriction [181]. Other studies have shown that Rho-kinase mRNA expression is downregulated in umbilical arteries of PE women [182], making it important to further investigate the role of Rho-kinase in the vascular changes associated with PE.

Circulating Biomarkers

Measurement of plasma levels of biomarkers such as VEGF, sFlt-1 and sEng might allow the stratification of PE patients in different categories according to the severity of symptoms and thus improve clinical management of the disease [194] (Table 5). Biomarkers also allow early disease assessment in asymptomatic pregnant women, in particular among target groups at increased risk based on their clinical history (PE or HTN in a previous pregnancy) or pre-pregnancy state (HTN, obesity, autoimmune disease).

Microarray analysis may help to screen the placental transcriptome for upregulated and downregulated genes in PE samples compared to healthy controls [195]. Studies have selected several target genes that are produced mainly by the placenta and that show increased protein concentrations in patients with PE. Based on the result, mRNA levels of plasminogen activator inhibitor-1 (SERPINE1), tissue-type plasminogen activator (PLAT), vascular endothelial growth factor (VEGFA), VEGFA receptor 1 (FLT1), endoglin, placenta-specific 1 (PLAC1) and selectin P (SELP) were assessed in the plasma of women with and without PE. These mRNA expressions were increased in plasma from pregnant women who later developed PE in comparison to Norm-Preg women. Also, analysis showed FLT1 as the marker with the highest detection rate and PLAC1 with the lowest detection rate (Table 5). The best multivariable model was obtained by the combination of all markers [196].

Despite the existence of many potential markers for PE, their reliability in predicting PE has not been consistent and the predictive value of different markers needs to be further assessed in order to identify the best marker combination for use in clinical settings.

Immune system

The status of the immune system can be useful in predicting the risk of PE in early pregnancy. The immune system fights off foreign pathogens partly through activation of the alternate complement pathway and the factor B-derived Bb fragment, a process that could also cause vascular injury and fetal demise. The maternal abnormal immune response to paternal antigens is prominent in PE. PE women show higher levels of Bb than Norm-Preg women [212]. Also, PE is more common during a first conception, after the mother has switched partners, in women who use barrier contraception, and in cases of donated gametes. Conversely, conditions associated with suppressed immune response such as HIV-related T-cell immune deficiency are associated with a low rate of PE [29].

An increase in inflammatory factors in PE may also represent an abnormal maternal immunological response, comprising a change in the role of monocytes and natural killer cells, altered release of circulating cytokines, and activation of pro-inflammatory AT₁R [29]. Neutrophils and other inflammatory cell profile could also be useful as biomarkers of PE. PE is characterized by leukocyte activation, which may account for the elevated cytokine levels found in the disease [213–216]. Therefore, it has been suggested that PE may represent an excess maternal inflammatory response to pregnancy [217]. In particular, it has been shown that maternal neutrophils are activated during their passage in the decidua of women with PE [216] and that neutrophil activation can be achieved by the conditioned medium of placental villous culture taken from PE pregnancies [218]. These findings further suggest that decidual/placental inflammatory factors are involved in the disease.

Platelet Count

The mean platelet volume (MPV) could be an important predictor of PE. Longitudinal studies have shown that women who develop PE have higher MPV 4.6 weeks prior to the appearance of symptoms when compared to Norm-Preg women [219]. Also, thrombocytopenia is a common abnormality in PE, and the degree of thrombocytopenia increases with the severity of the disease [9].

Uric Acid

Uric Acid is a marker of oxidative stress, tissue injury and renal dysfunction, and may be helpful in the prediction of PE [220]. During Norm-Preg, uric acid levels decrease initially, but then gradually increase over gestational time. Hypoxia and ischemia of the placenta and cytokines such as interferon induce the expression of xanthine oxidase and increase the production of uric acid and ROS [221]. During PE, hyperuricemia develops as early as 10 wk of gestation. Elevated levels of circulating uric acid may contribute to the pathogenesis of PE and may attenuate normal trophoblast invasion and spiral artery remodeling [222]. Elevated uric acid levels could contribute to the reduced production of NO and the altered endothelial function in PE women. Also, uric acid stimulates human monocytes to produce TNF-α, IL-6 and IL-1β, which are elevated in PE [222]. Recent meta-analysis further supported that measurement of serum uric acid may be a useful test to predict maternal complications in management of PE [223].

Amniotic Fluid

Amniotic fluid derived by amniocentesis in the second trimester may be helpful in the prediction of PE. In a recent study, amniotic fluid samples were tested for insulin-like factor 3 (INSL3), a member of the insulin/relaxin family of peptide hormones made by the fetal testis and responsible for the first trans-abdominal phase of testicular descent. The results demonstrated that, in male fetus only, INSL3 was elevated in asymptomatic women who subsequently developed PE [224]. Other studies showed that the levels of amniotic fluid inhibin A, a glycoprotein produced by syncytiotrophoblast, in pregnant women who subsequently developed severe PE were higher than in Norm-Preg women [225]. Also, the level of sFlt1 is higher in the amniotic fluid of PE (51,040 pg/ml) than Norm-Preg women (33.490 pg/ml) [226]. Thus, careful monitoring of the hormones in the amniotic fluid could be a potential indicator of PE.

Uterine artery Doppler screening

Uterine artery Doppler screening at 23 weeks of pregnancy may predict PE as an abnormal early diastolic bilateral uterine artery notching in the waveform. However, about 50% of mothers with uterine artery notching do not develop PE. Plasma levels of fibrinolytic markers such as tissue-type plasminogen activator (t-PA), PAI-1, PAI-2, plasmin-α2-antiplasmin (PAP) and D-dimers have been used in conjunction with uterine artery Doppler screening as predictors of PE. Uterine artery notching at 23 weeks of gestation is associated with increased PAI-1 levels. Within the uterine artery notching group, mothers who developed PE had increased t-PA levels and decreased PAI-2 levels. Thus while no single fibrinolytic marker may be helpful in determining pregnancy outcome in women with uterine artery notching, t-PA and PAI-2 are worthy of study [227].