Rabbany SY, Heissig B, Hattori K & Rafii S: Urbich C & Dimmeler S: 2004. Endo- modulates signaling from the G-CSF recep- 2003. Molecular pathways regulating mobi- thelial progenitor cells functional char- tor. Leukemia 14:1284–1291. lization of marrow-derived stem cells for acterization. Trends Cardiovasc Med 14: You-Ten KE, Muise ES, Itie A, et al.: 1997. – tissue revascularization. Trends Mol Med 9: 318 322. Impaired bone marrow microenvironment – 109 117. Vercauteren M, Remy E, Devaux C, et al.: 2006. and immune function in T cell Ribatti D: 2008. Hemangioblast does exist. Improvement of peripheral endothelial dys- tyrosine phosphatase-deficient mice. J Exp Leuk Res 32:850–854. function by protein tyrosine phosphatase Med 186:683–693. inhibitors in heart failure. Circulation 114: Salmond RJ & Alexander DR: 2006. SHP2 YuM,LuoJ,YangW,etal.:2006.The 2498–2507. forecast for the immune system: fog gra- scaffolding adapter Gab2, via Shp-2, reg- dually clearing. Trends Immunol 27: Walter DH, Haendeler J, Reinhold J, et al.: ulates kit-evoked mast cell proliferation by 154–160. 2005. Impaired CXCR4 signaling contri- activating the Rac/JNK pathway. J Biol butes to the reduced neovascularization Chem 281:28615–28626. Seki Y, Kai H, Shibata R, et al.: 2000. Role of capacity of endothelial progenitor cells the JAK/STAT pathway in rat carotid artery from patients with coronary artery disease. remodeling after vascular injury. Circ Res Circ Res 97:1142–1151. 87:12–18. Ward AC, Oomen SP, Smith L, et al.: 2000. The Sharlow ER, Pacifici R, Crouse J, et al.: 1997. SH2 domain-containing protein tyrosine Hematopoietic cell phosphatase negatively phosphatase SHP-1 is induced by granulo- regulates erythropoietin-induced hemoglo- cyte colony-stimulating factor (G-CSF) and PII S1050-1738(08)00101-1 TCM binization in erythroleukemic SKT6 cells. Blood 90:2175–2187. Shepherd RM, Capoccia BJ, Devine SM, et al.: 2006. Angiogenic cells can be rapidly mobi- lized and efficiently harvested from the blood following treatment with AMD3100. Blood 108:3662–3667. Simoncic PD, Bourdeau A, Lee-Loy A, et al.: 2006. T-cell protein tyrosine phosphatase (Tcptp) is a negative regulator of colony- Novel Biomarkers for Predicting stimulating factor 1 signaling and macro- phage differentiation. Mol Cell Biol 26: Preeclampsia – 4149 4160. David M. Carty, Christian Delles, and Anna F. Dominiczak⁎ Socinski MA, Cannistra SA, Elias A, et al.: 1988. Granulocyte–macrophage colony sti- mulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1:1194–1198. Preeclampsia is a major cause of maternal morbidity and mortality Sugano M, Tsuchida K, Maeda T & Makino N: worldwide. Despite decades of research into the condition, the ability of 2007. SiRNA targeting SHP-1 accelerates clinicians to predict preeclampsia prior to the onset of symptoms has not angiogenesis in a rat model of hindlimb improved significantly. In this review, we will examine the pathophysiol- ischemia. Atherosclerosis 191:33–39. ogy underlying preeclampsia and will look at potential biomarkers for Takahashi T, Kalka C, Masuda H, et al.: 1999. Ischemia- and cytokine-induced mobiliza- early prediction and diagnosis. In addition, we will explore potential tion of bone marrow-derived endothelial future areas of research into the condition. (Trends Cardiovasc Med progenitor cells for neovascularization. Nat – n Med 5:434–438. 2008;18:186 194) 2008, Elsevier Inc. Tan Y, Shao H, Eton D, et al.: 2007. Stromal cell-derived factor-1 enhances pro-angio-  Introduction a major cause of maternal morbidity and genic effect of granulocyte-colony stimulat- mortality worldwide. The cardinal clin- ing factor. Cardiovasc Res 73:823–832. Preeclampsia is a multisystem disorder ical features of the condition are hyper- of pregnancy, which complicates 3%-5% Thum T, Hoeber S, Froese S, et al.: 2007. Age- tension and proteinuria occurring after dependent impairment of endothelial pro- of pregnancies in the western world. It is genitor cells is corrected by growth-hor- 20 weeks gestation in women who were mone-mediated increase of insulin-like not previously known to be hypertensive. growth-factor-1. Circ Res 100:434–443. Other signs and symptoms include David M. Carty, Christian Delles, and Anna F. edema and headache, and in severe Tsui FW, Martin A, Wang J & Tsui HW: 2006. Dominiczak are at the BHF Glasgow Cardio- Investigations into the regulation and func- vascular Research Centre, University of Glas- cases, the condition is associated with tion of the SH2 domain-containing protein- gow, G12 8TA Glasgow, United Kingdom. seizures (eclampsia), liver, and kidney tyrosine phosphatase, SHP-1. Immunol Res ⁎ Address correspondence to: Prof. Anna F. dysfunction as well as clotting abnorm- – 35:127 136. Dominiczak, BHF Glasgow Cardiovascular alities, Adult Respiratory Distress Syn- Urbich C, Aicher A, Heeschen C, et al.: 2005. Research Centre, University of Glasgow, 126 drome and fetal growth restriction (FGR) Soluble factors released by endothelial University Place, G12 8TA Glasgow, United (Davison et al. 2004). progenitor cells promote migration of Kingdom. Tel.: (+44) 141 330 5420; fax: (+44) endothelial cells and cardiac resident pro- 141 330 6997; e-mail: [email protected]. The cause of preeclampsia remains genitor cells. J Mol Cell Cardiol 39: © 2008 Elsevier Inc. All rights reserved. unknown, and the only known cure is 733–742. 1050-1738/08/$-see front matter delivery of the fetus and placenta.

186 TCM Vol. 18, No. 5, 2008 Various agents have been evaluated to The imaging technique that has so far ments of the spiral arteries, at around 10- see whether they influence the develop- been most widely used for predicting 12 weeks gestation; the second involves ment of preeclampsia. Aspirin has been preeclampsia has been uteroplacental invasion of the myometrial segments at studied extensively; it was summarized Doppler ultrasound. Impaired placental 15-16 weeks (Sargent and Smarason in a Cochrane review by Duley et al. perfusion, one of the hallmarks of pre- 1995). In preeclampsia, the cytotropho- (2004) to have modest but significant eclampsia, can be assessed by measuring blastic invasion of the myometrial seg- benefits in preventing preeclampsia. It is flow waveform ratios or by detecting ments is impaired: the spiral arteries commonly used in women who are diastolic notching of the uterine arcuate remain narrow, and blood supply to the known on the basis of risk factors to be vessels. Uterine Doppler studies can, fetus is restricted. The effects of this on at high risk of developing preeclampsia, however, be inconsistent: different types the fetus become more significant as although questions remain over exactly of machine, different gestational age at pregnancy progresses, since the uterine which subgroups of women are most assessment, and different definitions of vasculature is unable to keep up with the likely to benefit. Studies of calcium abnormal flow velocity waveforms make increased amount of blood and nutrients supplementation suggest that it is of accurate comparison of studies difficult. necessary for fetal development. Placen- most benefit when used in high-risk A large systematic review of 43 studies tal ischemia is thought to develop as a women with low dietary calcium intake involving 40,000 patients (Conde-Agu- result of this abnormal cytotrophoblastic (Hofmeyr et al. 2006), although studies of delo et al. 2004) found that, in both low- invasion; this has been proposed as antioxidants such as vitamins C and E and high-risk patient groups, the positive leading to release of placental factors and magnesium and zinc supplements predictive value was not sufficiently high and imbalance of angiogenic factors, have been less promising (Poston et al. to recommend routine screening. causing the widespread endothelial dys- 2006). Attention has therefore turned in function which characterizes preeclamp- Despite decades of research into the recent years towards identifying mater- sia (Lee et al. 2007). condition, predicting which women are at nal markers of placental dysfunction increased risk of developing preeclampsia which are raised in women who go on  Angiogenic factors remains problematic. Identifying “at-risk” to develop preeclampsia. In this review, women is an important aim; because we will look at novel biomarkers which As research in the field of preeclampsia modern obstetric care places emphasis have been used in the prediction of progresses, much of the attention in upon the primary care setting for expec- preeclampsia and explore potential recent years has been focused on peptides tant women, a marker which identified future areas of investigation. related to angiogenesis. Angiogenesis, the high-risk women would allow for closer development of new blood vessels from supervision in secondary care. Such a existing endothelium, is essential for  Pathogenesis of preeclampsia marker would also facilitate recruitment normal placental development. Two of for trials of potential therapeutic agents, Although the cause of preeclampsia the angiogenic growth factors, vascular for accurate diagnosis, and for timely remains elusive, the origin of the condi- endothelial growth factor (VEGF) and intervention whenever problems develop. tion is recognized as lying in the placenta. placental growth factor (PlGF) are Furthermore, predicting preeclampsia in This is known to be the case because thought to contribute to normal tropho- women with underlying conditions such preeclampsia occurs only in the presence blastic proliferation and implantation as diabetes and chronic hypertension of pregnancy, it resolves after delivery of (Thadhani et al. 2004), and it has been would be of great clinical value. the placenta and it can occur in the hypothesized that an imbalance in levels Clinicians have traditionally relied on absence of a viable fetus, for example, in of these growth factors has a crucial role maternal risk factors, such as increased molar pregnancies. Placental develop- in preeclampsia. As normal pregnancy maternal age, family history, and preex- ment is a closely regulated process which progresses, maternal VEGF expression is isting diseases, for determining which is essential for normal fetal development reduced (Ong et al. 2000a), but placental women are at increased risk. The pro- and for maintaining a successful preg- levels of mRNA-encoding VEGF have blem with using these risk factors is that nancy. Blood supply to the placenta is via been shown to be much lower in women millions of women worldwide have these the spiral arteries, which, in turn, are with preeclampsia compared to controls risk factors but do not develop pree- branches of the uterine arteries. Early in (Cooper et al. 1996). Similarly, maternal clampsia. Moreover, the majority of normal pregnancy, the cytotrophoblastic plasma levels of PlGF have been shown to them are nonmodifiable. There have cells of the developing placenta invade the be significantly reduced in the second been many screening tests evaluated in uterine wall, disrupting the endothelium trimester in women who went on to the literature over the years for predict- and tunica media of the spiral arteries. The develop preeclampsia compared to con- ing preeclampsia; these have been com- vascular wall of the spiral arteries is trols (Kim et al. 2007). The use of anti- prehensively reviewed in a World Health remodeled; this, in turn, leads to a trans- VEGF for systemic treatment Organization publication (Conde-Agu- formation of the spiral arteries from low- in cancer has shown a dose dependant delo et al. 2004)(Figure 1). A further flow, highly resistant vessels into the high- association with hypertension and pro- summary of predictive tests, including a flow, low-resistance vessels which are vital teinuria (Ostendorf et al. 2007), which review of preventative interventions and for normal placental development. may indicate that these factors have a role economic modeling, has been recently There are thought to be two stages to in the development of preeclampsia. published by the UK National Institute cytotrophoblastic invasion: the first Many recent studies have therefore con- for Health Research (Meads et al. 2008). involves invasion of the decidual seg- centrated on factors which antagonize

TCM Vol. 18, No. 5, 2008 187 VEGF and PlGF to assess their role in the  Soluble Endoglin but abnormally low levels of PP-13 have development of preeclampsia. Two of the been shown in weeks 11-13 gestation in Another peptide that has been implicated most extensively studied peptides, which women who went on to develop pree- in the pathogenesis of preeclampsia is are produced by the placenta, are soluble clampsia and FGR when compared with soluble endoglin (sEng). Endoglin, a FMS-like tyrosine kinase (sFLT-1) and controls (Burger et al. 2004). A further coreceptor for transforming growth fac- soluble endoglin. study, analyzing maternal serum PP-13 tors β1 and β3(Luft 2006), is highly levels at 9-12 weeks gestation, also found expressed on endothelial cell membranes lower levels in women who went on to  Soluble sFLT-1 and syncytiotrophoblasts. Mutations in develop preeclampsia compared with the encoding Eng are the under- Soluble FLT-1 (also known as soluble controls (Chafetz et al. 2007). Combining lying cause of hereditary haemorrhagic VEGF receptor 1 or sFLT-1) is a secreted maternal serum PP-13 to uterine artery telangiectasia, a genetic condition char- splice variant of FLT-1. It binds to and Doppler studies early in pregnancy seems acterized by atrioventricular malforma- neutralizes the angiogenic actions of to improve ability to predict severe tions, epistaxis, and telangiectasiae VEGF and PlGF (Venkatesha et al. 2006) preeclampsia. Nicolaides et al. (2006) (Venkatesha et al. 2006). In normal and is thought to be one of the key found that women who went on to pregnancy, levels of sEng fall between peptides involved in the development of develop preterm preeclampsia (requiring the first and second trimesters, but in preeclampsia. Maternal serum levels of delivery before 34 weeks) had a higher women who go on to develop preeclamp- sFLT-1 have been shown to be elevated in median uterine artery pulsatility index, sia, this reduction is blunted (Rana et al. women with preeclampsia compared to and a lower median serum PP-13 in the 2007). Consistent with studies involving controls (Koga et al. 2003, Salahuddin et first trimester when compared to con- sFLT-1, it has also been demonstrated al. 2007, Chaiworapongsa et al. 2004), to trols. Thus, they concluded that, for a that levels of sEng are elevated in the correlate with disease severity (Chaiwor- 90% detection rate of the condition, sera of pregnant women with preeclamp- apongsa et al. 2004) and to decrease using serum PP-13 for all women and sia, correlate with disease severity, and markedly following delivery (Koga et al. Doppler studies in the 14% at highest fall after delivery (Venkatesha et al. 2003). It has also been shown that that risk, a false-positive rate of 6% could be 2006). A promising discovery in terms levels of sFLT-1 are increased in nullipar- achieved. PP-13, therefore, whether used of predicting the condition was that ity (one of the main known risk factors for alone or in combination with Doppler levels of sEng are elevated several developing preeclampsia) when com- studies, appears to be a promising area weeks before the development of clinical pared to multiparous women (Wolf et al. for future research in this field. symptoms in women who developed 2005). A series of studies by Maynard et preeclampsia; furthermore, in patients al. (2003) revealed that mRNA of sFLT-1 who developed preterm preeclampsia,  Pregnancy-Associated Plasma is up-regulated in the placenta of women the serum sEng levels are elevated Protein A with preeclampsia, leading to increased (approximately twofold) as early as systemic levels. Furthermore, these Pregnancy-associated plasma protein A, gestational weeks 17-20 (Levine et al. authors demonstrated that when a (PAPP-A) is a large highly glycosylated 2006). Rana et al. (2007) found that recombinant adenovirus encoding sFLT- protein complex produced by the devel- although levels of sEng and sFLT-1 1 was injected into pregnant rats, hyper- oping trophoblast (Bersinger et al. 2003), were found to be elevated in the serum tension and proteinuria, as well as glo- whichisusedinmanycentersasa of preeclamptic women at 17-20 weeks merular endotheliosis, one of the typical marker for Downs' syndrome. It has gestation when compared to controls, pathological lesions seen in preeclamp- been shown to be responsible for the the levels at 11-13 weeks were similar sia, were observed. Further evidence for cleavage of insulin-like growth factor between cases and controls. Both sEng the placental origin of the elevated sFLT-1 (IGF) binding , which are inhibi- and sFLT-1 appear, therefore, to be was provided by Staff et al. (2005). Given tors of IGF action, in several biological important peptides in the pathogenesis that “delivery” includes both the fetus and fluids (Laursen et al. 2001). PAPP-A was of preeclampsia although, when used the placenta, the group investigated first shown to be elevated in the plasma of alone, do not appear to have a suffi- whether fetal as well as maternal levels preeclamptic women nearly 30 years ago ciently high positive predictive value to of sFLT-1 were elevated in preeclampsia. (Hughes et al. 1980). More recent studies be translated into routine clinical practice. They found that although fetal levels of have shown that although reduced first sFLT-1 (measured in cord blood) are trimester serum levels of PAPP-A are elevated in preeclampsia, the maternal associated with preeclampsia, levels are  Placental Protein 13 serum levels were 29-fold higher and also low in other complications of preg- concluded that there was no substantial Placental protein 13 (PP-13) is a 32-kDa nancy (Ong et al. 2000b, Yaron et al. 2002, fetal contribution to the elevated circulat- dimer protein, one of a group of proteins Smith et al. 2002). It has been suggested ing maternal sFLT-1 levels seen in pre- which are known to be highly expressed that PAPP-A is more useful as a marker of eclampsia. Finally, serum sFLT-1 levels in the placenta. It has been prepared in FGR than of preeclampsia (Canini et al. have been shown to be increased in recombinant form and is thought to be 2008). In a recent paper, Spencer et al. women with preeclampsia superimposed involved in placental implantation and (2008) described a small increase in upon systemic lupus erythematosis (Qazi maternal vascular remodeling (Nico- likelihood ratio of developing preeclamp- et al. 2008) and glomerulonephritis laides et al. 2006). During normal preg- sia with decreasing levels of PAPP-A. (Masuyama et al. 2006). nancy, levels of PP-13 gradually increase, Although PAPP-A alone was not a good

188 TCM Vol. 18, No. 5, 2008 predictor for preeclampsia, they felt, increased insulin sensitivity, there fol- ing at SHBG levels at weeks 10-14 in 107 similarly to PP-13, that sensitivity could lows a progressive increase in insulin women with preeclampsia found no be improved by combining with uterine resistance throughout the second and significant difference between women artery Doppler studies. third trimesters (Butte 2000). Sex-hor- who went on to develop preeclampsia mone-binding (SHBG) is a compared to controls (Spencer et al. produced by the liver 2005). This retrospective study included  Insulin Resistance which binds circulating estrogens and multiparous women, which may have Insulin resistance has long been impli- testosterone. Production of SHBG is confounded results. Another study found cated in the pathogenesis of preeclamp- inhibited by insulin; therefore, low levels no difference at gestational weeks 17 or sia. Carbohydrate metabolism is known of SHBG are associated with elevated 33 between 29 cases of preeclampsia and to be altered in women with preeclamp- insulin levels. As a result, studies have controls (Carlsen et al. 2005), although sia, whereas fasting insulin levels have used low SHBG levels as a marker of the relatively small numbers of patients also been shown to be elevated prior to insulin resistance in both cardiovascular involved may be of relevance. the onset of disease (Spencer et al. disease (Sherif et al. 1998)andin Adiponectin, an adipocyte-derived 2005). Furthermore, types 1 and 2 preeclampsia (Carlsen et al. 2005, Spen- cytokine involved in carbohydrate and diabetes, gestational diabetes, and poly- cer et al. 2005, Wolf et al. 2002). One fat metabolism, is another protein whose cystic ovarian syndrome are all well- study looking at first-trimester SHBG levels are inversely correlated with insu- established risk factors for the condition levels in 45 nulliparous women who lin resistance. High concentrations of (Wolf et al. 2002). developed preeclampsia found that levels adiponectin have been shown to be Normal pregnancy is characterized by were significantly reduced when com- protective against the development of increased insulin secretion by the pan- pared with controls (Wolf et al. 2002). In type 2 diabetes (Lindsay et al. 2002), creatic β cells, and, following initially contrast, however, a further study look- and serum levels of adiponectin have

Figure 1. Biomarkers of pre-eclampsia. Biomarkers of preeclampsia are grouped into four major categories (modified from Conde-Agudelo et al. 2004). Production and levels of biomarkers are ultimately dependent on genetic factors and therefore genomic studies are likely to detect genetic variants associated with preeclampsia. However, in contrast to the static genome, the proteome is dynamic. Whereas the genome will not change during pregnancy or pregnancy-associated conditions such as preeclampsia, the proteome will change. This is indicated by the double-headed arrow. Proteomic and metabolomic studies will therefore reflect a large number of biomarkers and their actual levels and will more accurately predict risk than genomic studies.

TCM Vol. 18, No. 5, 2008 189 been shown to correlate with sEng levels with an increased risk of preeclampsia. preeclampsia. Both are , in women with preeclampsia (Masuyama Nagy et al. (1998), found a higher are members of the transforming et al. 2007). It has been hypothesized that incidence of the ApoE e2 allele amongst growth factor β family, and during low serum adiponectin levels are asso- women with preeclampsia compared to pregnancy, are largely released by the ciated with an increased risk of develop- controls. Makkonen et al. (2001) studied fetoplacental unit. Inhibin A has an ment of preeclampsia. D'Anna et al. 133 women with preeclampsia and, in important endocrine role in the negative (2006) examined serum adiponectin contrast, found that none of the ApoE feedback of gonadotrophins, whereas levels in the first trimester in women alleles were over represented when com- activin A is thought to have activity in who subsequently developed preeclamp- pared with controls, findings that have various biological tissues (Luisi et al. sia. They found that levels were lower been confirmed elsewhere (Belo et al. 2005, Muttukrishna et al. 2000). In than those in controls, but the levels were 2004). The role of this avenue of investi- normal pregnancy, concentrations of significantly different between those who gation in the prediction of preeclampsia both hormones rise in the third trime- developed early-onset and late-onset is therefore, at present, uncertain. ster, and levels have been shown to be symptoms, suggesting a different patho- elevated approximately 10-fold in genesis. Ramsay et al. (2003) found, in women with severe preeclampsia com-  Inhibin A and Activin A contrast to their initial hypothesis, that pared to controls (Muttukrishna et al. serum adiponectin levels in the third Many studies have been reported using 1997). Second trimester levels of inhibin trimester are in fact higher in women inhibin A and activin A as predictors of A have been shown to be elevated in with preeclampsia compared with con- trols, a finding which has been con- firmed elsewhere (Fasshauer et al. 2008). It has been suggested that adipo- nectin forms part of the physiological response to preeclampsia, by improving insulin sensitivity (Fasshauer et al. 2008). It appears, therefore, that although insu- lin resistance has a prominent role in the pathogenesis of preeclampsia, its role in predicting the disease, and the best method for measuring it, are yet to be determined.

 Apolipoprotein E

One of the mechanisms by which pre- eclampsia has been postulated to develop is via abnormal lipid metabolism asso- ciated with oxidative stress. Women with preeclampsia have an abnormal lipid profile, with elevated concentrations of triglyceride-rich lipoproteins, which may contribute to endothelial dysfunction (Sattar et al. 1997). Apolipoprotein E (ApoE) is a major constituent of very low-density lipoproteins (VLDLs) whose role involves modifying inflammatory responses, and removal of excess choles- terol from the circulation via regulation of hepatic uptake (Belo et al 2004). The APOE gene on chromosome 19 has 3 Figure 2. Capillary electrophoresis online coupled to mass spectrometry. Urine samples are common alleles, encoding 3 plasma prepared for analysis, polypeptides are separated by capillary electrophoresis and directly ApoE isoforms, e2, e3, and e4. ApoE e4 sprayed into electrospray ionization–time of flight mass spectrometry. Data are evaluated using is a known risk factor for familial specific software solutions. Each polypeptide is defined by its accurate mass and normalized CE Alzheimer's disease, whereas both e2 migration time. Signal intensity serves as measure of the relative abundance. The data are and e4 isoforms have been associated stored as peak lists summarizing the information in a database. The process is demonstrated for use of CE–mass spectrometry to in the diagnosis of coronary artery disease. The lower panel with abnormally high triglyceride and shows a coronary artery disease-specific polypeptide pattern. The top panel is modified with VLDL levels (Francoual et al. 2002). It permission from Electrophoresis 2007;28:1407-1417. Sniehotta et al: CE - a multifunctional has been postulated that ApoE levels and application for clinical diagnosis. Electrophoresis. 2007. Volume 28. Pages 1407-1417. polymorphisms of its gene are associated Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

190 TCM Vol. 18, No. 5, 2008 both serum (Aquilina et al. 1999) and Further advances are expected from of these mechanisms can be integrated. amniotic fluid (Kim et al. 2006)in proteomic research. Proteomics has been In addition, since proteomics is a women who went on to develop severe defined as “knowledge of the structure, “hypothesis-free” research tool, it is likely preeclampsia, and when measured at function, and expression of all proteins in that this line of investigation will open term, serum levels have been shown to the biochemical or biological contexts of new avenues for potential biomarker correlate with preeclampsia severity all organisms” (Kenyon et al. 2002). discovery and for new diagnostic and (Zeeman et al. 2002). Interestingly, Comparing protein patterns between preventative measures. urinary activin A and inhibin A levels healthy patients and those with a disease have also been found to be elevated in has been increasingly used in recent  Metabolomics women with preeclampsia, as have years to discover markers of disease uterine vein levels (Muttukrishna et al. (biomarkers), which have a number of Metabolomics is a further area that has 2006). The second trimester levels of important roles in medical research. the potential to contribute significantly both inhibin A and activin A have been Proteomics can be used to improve to future research in preeclampsia. In reported to add significant prognostic early detection of disease, to develop complement to studies examining the information when measured in women new targets for therapeutic treatment, human genome and proteome, metabo- with abnormal uterine artery Doppler and to monitor response to treatment lomics can be defined as a “systematic studies (Florio et al. 2003). In contrast (Figure 1). To date, potential proteomic study of the unique chemical finger- to the above, however, a study by biomarkers have been reported for early prints that specific cellular processes Davidson et al. (2003) found that diagnosis of cardiovascular disease leave behind” (Daviss 2005). In common although second trimester levels of (Zimmerli et al. 2008), renal transplant with proteomics, studies of the human activin A were elevated in women who rejection (Wittke et al. 2005), urological metabolome can be carried out on went on to develop preeclampsia, inhi- cancers (Saito et al. 2005), and acute routine samples of urine, plasma, or bin A levels were not elevated, findings kidney injury (Zhou et al. 2006). Human serum requiring minimal specialist pre- confirmed by D'Anna et al. (2002). urine is an ideal medium to study paration of samples. Metabolomics (also Studies using first trimester inhibin A proteomics because of its ease of collec- referred to as metanomics or metabo- (Roes et al. 2004) have also shown a low tion, and its relative stability. Normal nomics) has been used to characterize predictive value. human urine contains up to 150 mg of signature profiles for cardiovascular The cause of these rises in activin A protein/24 hours and contains much disease (Brindle et al. 2002), for Alzhei- and inhibin A is not yet fully understood; useful information about the kidneys mer's disease (Han et al. 2002), and for and whether these hormones have a role and urogenital tract; because urine is a hypertension (Brindle et al. 2003). A in the etiology of preeclampsia is not yet filtrate of blood, pathological changes in study evaluating the ability of metabo- known. Further prospective studies, the blood indicative of disease can be lomics in diagnosis of preeclampsia has including measurement of urinary inhi- reflected in the urinary proteome (Gon- been reported (Kenny et al. 2005). They bin A, are clearly required. zalez-Buitrago et al. 2007). Proteomics discovered three metabolomic peaks employs protein separation using two- which could be used with high sensitiv- dimensional gel electrophoresis, high- ity and specificity to differentiate  Genomics and Proteomics performance liquid chromatography, or women with preeclampsia from con- A number of gene polymorphisms have capillary electrophoresis (CE), coupled trols. In this study, the majority of been found to be associated with the online to a time-of-flight mass spectro- samples were taken in late pregnancy, risk of developing preeclampsia. A meter (Figure 2). This allows the mole- and so, the ability of these metabolomic recent well-powered study into the cular weight of a single molecule to be peaks to predict preeclampsia before the genetics of preeclampsia, however, did measured and displayed according to its onset of clinical disease remains uncer- not confirm significant associations mass: charge ratio. Each peptide has a tain. Although the group were, at that between a single nucleotide polymorph- molecular “fingerprint,” and this tech- point, unable to identify the metabolites isms in candidate and preeclamp- nology allows simultaneous examination involved, it is likely that future research sia (GOPEC Consortium 2005). Recent of several thousand peptides within in this field will aid our understanding advances in genotyping technology will minutes. Once a biomarker “signature of this challenging condition. facilitate genome-wide association stu- profile” has been identified, it can be dies in preeclampsia which will very compared with healthy closely matched  Conclusion likely result in novel candidate genes for controls, allowing for a disease-specific the disorder. Results from studies into biomarker to be identified. Although improvements in obstetric and other polygenic disorders such as cor- It appears likely that the development neonatal care have led to a reduction in onary artery disease and diabetes are of preeclampsia involves several different morbidity and mortality from pree- promising (Wellcome Trust Case Con- pathophysiological mechanisms, as evi- clampsia, our ability to predict the con- trol Consortium 2007), but investigators denced by the diversity of the peptides dition has not improved significantly. We will have to follow strict rules to avoid that have been studied to date. Current currently rely on “secondary prevention” false positive and underpowered nega- research is driven by pathways that are of preeclampsia: women who have pre- tive results (NCI-NHGRI Working known to be involved in the development viously had the disease are closely mon- Group on Replication in Association of the condition, and one advantage of itored throughout pregnancy. The Studies 2007). proteomics-based research is that many majority of women who develop

TCM Vol. 18, No. 5, 2008 191 preeclampsia, however, are only diag-  Acknowledgment Chaiworapongsa T, Romero R, Espinoza J, nosed once they have developed the full- et al.: 2004. Evidence supporting a role for This work has been supported by the blown manifestations of the condition, blockade of the vascular endothelial British Heart Foundation Chair and Pro- growth factor system in the pathophysiol- by which time treatment options are gramme Grant BHF RG/07/005/23633, the ogy of preeclampsia—Young Investigator limited. Rather than being a separate European Union's Sixth Framework Pro- Award. Am J Obstet Gynecol 190: condition, preeclampsia has been pre- 1541–1547. gramme “InGenious HyperCare” LSHM- viously described as the extreme end of a CT-2006-037093, and the Scottish Funding Conde-Agudelo A, Villar J & Lindheimer M: maternal systemic response engendered 2004. World Health Organization systema- Council Strategic Research Development by pregnancy itself (Redman and Sargent tic review of screening tests for preeclamp- Grant “Biomarkers for Battling Chronic 2003). We have seen that many of the sia. Obstet Gynecol 104:1367–1391. Disease” HR07001. proposed biomarkers for preeclampsia Cooper JC, Sharkey AM, Charnock-Jones DS, are raised to a lesser extent in normal et al.: 1996. VEGF mRNA levels in placentae pregnancy, which will make discovery of References from pregnancies complicated by pre- accurate biomarkers for preeclampsia eclampsia. Br J Obstet Gynaecol 103: Aquilina J, Barnett A, Thompson O & Har- 1191–1196. more difficult. Nevertheless, if signs of rington K: 1999. Second-trimester maternal D'Anna R, Baviera G, Corrado F, et al.: 2006. abnormal placental and endothelial dys- serum inhibin A concentration as an early Adiponectin and insulin resistance in early- function could be detected prior to the marker for preeclampsia. 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Maternal serum activin, inhibin, increased serum levels of pregnancy-asso- human chorionic gonadotrophin and most of which have a far lower incidence ciated plasma protein a (PAPP-A), inhibin A, alpha-fetoprotein as second trimester pre- than preeclampsia, and it is hoped that activin A, and soluble E-selectin. Hypertens dictors of pre-eclampsia. Br J Obstet Gynae- – in time we will be able to do the same Pregnancy 22:45 55. col 110:46–52. Brindle JT, Antti H, Holmes E, et al.: 2002. for preeclampsia. Davison JM, Homuth V, Jeyabalan A, et al.: An increased understanding of the Rapid and noninvasive diagnosis of the 2004. New aspects in the pathophysiology of molecular mechanisms underlying pre- presence and severity of coronary heart Preeclampsia. J Am Soc Nephrol 15: eclampsia has led to several potential disease using H-1-NMR-based metabo- 2440–2448. nomics. Nature Med 8:1439–1444. areas of investigation. In addition to Daviss B: 2005. Growing pains for metabolo- Brindle JT, Nicholson JK, Schofield PM, et al.: studies mentioned above, novel biomar- mics. Scientist 19:25–28. 2003. Application of chemometrics to H-1 kers such as urine orosomucoid, an acute NMR spectroscopic data to investigate a Duley L, Henderson-Smart DJ, Meher S & phase protein, show promise in early relationship between human serum meta- King JF: 2004. Antiplatelet agents for pre- prediction of preeclampsia (Kronborg et bolic profiles and hypertension. Analyst 128: venting pre-eclampsia and its complications al. 2007). To date, however, studies of 32–36. (Review). Cochrane Database Syst Rev CD004659. potential biomarkers for predicting pre- Burger O, Pick E, Zwickel J, et al.: 2004. eclampsia have involved relatively small Placental protein 13 (PP-13): Effects on Fasshauer M, Waldeyer T, Seeger J, et al.: numbers of patients, although many of cultured trophoblasts, and its detection in 2008. 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TCM Vol. 18, No. 5, 2008 193 Spencer K, Cowans NJ & Nicolaides KH: 2008. Low levels of maternal serum PAPP-A in the first trimester and the risk of pre-eclampsia. Prenat Diagn 28:7–10. Spencer K, Yu CKH, Rembouskos G, et al.: 2005. First trimester sex hormone-binding Of Cardiovascular Illness and Diversity globulin and subsequent development of preeclampsia or other adverse pregnancy of Biological Response outcomes. Hypertens Pregnancy 24:303–311. Pascal J. Goldschmidt-Clermont⁎, Chunming Dong, Staff AC, Braekke K, Harsem NK, et al.: 2005. Mike West, and David M. Seo Circulating concentrations of sFlt1 (soluble fins-like tyrosine kinase 1) in fetal and maternal serum during pre-eclampsia. Eur – J Obstet Gynecol Reprod Biol 122:33 39. Noise in gene expression (stochastic variation in the composition of the Thadhani R, Mutter WP, Wolf M, et al.: 2004. 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J Clin loss of diversity in the organism's response. (Trends Cardiovasc Med Endocrinol Metabol 87:1563–1568. 2008;18:194–197) n 2008, Elsevier Inc. Wolf M, Shah A, Lam C, et al.: 2005. Circulat- ing levels of the antiangiogenic marker  Noise in Gene Expression— variation in gene expression among cells sFLT-1 are increased in first versus second A Requisite for Health? and organisms within a homogeneous pregnancies. Am J Obstet Gynecol 193: population in common environment. The 16–22. Over the past couple of years, substantial potential sources for such a variation Yaron Y, Heifetz S, Ochshorn Y, et al.: 2002. progress has been made in the identifica- include (1) inherent stochasticity of Decreased first trimester PAPP-A is a pre- tion of genetic sources underlying biochemical processes involving infre- dictor of adverse pregnancy outcome. Pre- expression differences between indivi- nat Diagn 22:778–782. quent molecular events concerning dual cells and organisms (Raser and small numbers of molecules; (2) differ- Zeeman GG, Alexander JM, McIntire DD, O'Shea 2005). The differences (noise) in et al.: 2002. Inhibin-A levels and severity of ences in the internal states of a popula- gene expression refer to the stochastic hypertensive disorders due to pregnancy. tion of cells and organisms, such as Obstet Gynecol 100:140–144. chromatin modification (epigenetics); Zhou H, Pisitkun T, Aponte A, et al.: 2006. Pascal J. Goldschmidt-Clermont, Chunming (3) subtle environment differences, such Exosomal fetuin-A identified by proteo- Dong, and David M. Seo are at the University as morphogen gradients in multicellular mics: A novel urinary biomarker for detect- of Miami, Miller School of Medicine, Miami, development (such as regulatory small ing acute kidney injury. Kidney Int 70: FL 33136, USA. Mike West is at the Depart- RNA molecules); (4) ongoing genetic 1847–1857. ment of Statistical Science, Duke University, mutation; and (5) topology of gene Zimmerli LU, Schiffer E, Zürbig P, et al.: 2008. Durham, NC 27710, USA; and University of Urinary proteomic biomarkers in coronary Miami, FL 33136, USA. regulatory networks, such as presence ⁎ artery disease. Mol Cell Proteomics 7: Address correspondence to: Dr. Pascal J. and absence of functional feedback 290–298. Goldschmidt-Clermont, Miller School of Medi- loops. Although fluctuations in gene cine, Room 1143A Rosenstiel Medical Science expression as a result of intrinsic noise Building (R699), 1600 N.W. 10th Avenue, Uni- could be detrimental to cellular and versity of Miami, Miami, FL 33136, USA. Tel.: organismal physiology, they have been (+1) 305 243 6545; fax: (+1) 305 243 4888; e-mail: [email protected]. found to be beneficial in many circum- © 2008 Elsevier Inc. All rights reserved. stances. Indeed, noise has been shown to PII S1050-1738(08)00102-3 TCM 1050-1738/08/$-see front matter provide a mechanism for single cell

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