Preeclampsia, a disease of the maternal endothelium: the role of antiangiogenic factors and implications for later cardiovascular disease.

Abstract

HomeCirculationVol. 123, No. 24Preeclampsia, a Disease of the Maternal Endothelium Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBPreeclampsia, a Disease of the Maternal EndotheliumThe Role of Antiangiogenic Factors and Implications for Later Cardiovascular Disease Camille E. Powe, AB, Richard J. Levine, MD, MPH and S. Ananth Karumanchi, MD Camille E. PoweCamille E. Powe From Harvard Medical School, Boston, MA (C.E.P.); Department of Health and Human Services, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Division of Epidemiology, Statistics, and Prevention Research, Bethesda, MD (R.J.L.); and Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA (S.A.K.). Search for more papers by this author , Richard J. LevineRichard J. Levine From Harvard Medical School, Boston, MA (C.E.P.); Department of Health and Human Services, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Division of Epidemiology, Statistics, and Prevention Research, Bethesda, MD (R.J.L.); and Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA (S.A.K.). Search for more papers by this author and S. Ananth KarumanchiS. Ananth Karumanchi From Harvard Medical School, Boston, MA (C.E.P.); Department of Health and Human Services, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Division of Epidemiology, Statistics, and Prevention Research, Bethesda, MD (R.J.L.); and Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA (S.A.K.). Search for more papers by this author Originally published21 Jun 2011https://doi.org/10.1161/CIRCULATIONAHA.109.853127Circulation. 2011;123:2856–2869Preeclampsia is a clinical syndrome defined as the new onset of hypertension and proteinuria during the second half of pregnancy.1 It afflicts 3% to 5% of pregnancies and is a leading cause of maternal mortality, especially in developing countries.2,3 Because the only known remedy is delivery of the placenta, in developed countries preeclampsia is an important cause of premature delivery, usually medically indicated for the benefit of the mother. This results in infant morbidity and substantial healthcare expenditure.4 Despite the considerable morbidity and mortality, the cause of preeclampsia has remained enigmatic.Both hypertension and proteinuria implicate the endothelium as the target of the disease. The hypertension of preeclampsia is characterized by peripheral vasoconstriction and decreased arterial compliance.5,6 The proteinuria of preeclampsia is associated with a pathognomonic renal lesion known as glomerular endotheliosis, in which the endothelial cells of the glomerulus swell and endothelial fenestrations are lost.7,8 Podocyturia has been recently associated with preeclampsia during clinical disease9; however, whether this is the cause or effect of proteinuria is unknown. The glomerular filtration rate is decreased compared with normotensive pregnant women; in rare cases, acute renal failure may develop.Preeclampsia is a systemic vascular disorder that may also affect the liver and the brain in the mothers. When the liver is involved, women may present with abdominal pain, nausea, vomiting, and elevated liver enzymes. Pathological examination of the liver reveals periportal and sinusoidal fibrin deposition and, in more extreme cases, hemorrhage and necrosis.10 The severe preeclampsia variant HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) occurs in ≈20% of women with severe preeclampsia,11 and is named not only for the liver involvement, but also for the disorder of the coagulation system that develops.12 Approximately 20% of women with HELLP syndrome develop disseminated intravascular coagulation, which carries a poor prognosis for both mother and fetus.11 Placental abruption, ascites, hepatic infarction, hepatic rupture, intra-abdominal bleeding, pulmonary edema, and acute renal failure are all severe clinical manifestations associated with preeclampsia that can result in maternal death.13 Perhaps the most feared complication of preeclampsia is eclampsia itself, defined by the presence of seizures, for which women with severe preeclampsia are often treated with magnesium sulfate prophylaxis.1 The brain injury in eclampsia is associated with cerebral edema and characteristic white matter changes of reversible posterior leukoencephalopathy syndrome, which is similar to findings noted in hypertensive encephalopathy and with cytotoxic immunosuppressive therapies.14 Cerebrovascular complications, including stroke and cerebral hemorrhage, are responsible for the majority of eclampsia-related deaths.15 Complications affecting the developing fetus include indicated prematurity,16 intrauterine fetal growth restriction, oligohydramnios, bronchopulmonary dysplasia,17 and increased risk of perinatal death.18The risk factors for preeclampsia are varied and unique to this condition. Genetic factors are at least partially responsible, because both a maternal and a paternal family history of the disease predispose to preeclampsia.19 There is a 7-fold risk of recurrence for women who have had the disease in a previous pregnancy.20 Multiple gestation is an additional risk factor, and triplet gestation carries a greater risk than twin, suggesting that increased placental mass plays some role.20 Associations between preeclampsia and nulliparity,20 change in paternity from a previous pregnancy,21 increased interpregnancy interval,22 use of barrier contraception,23 and conception by intracytoplasmic sperm injection24 implicate limited recent exposure to paternal antigen as a predisposing factor. Notably, classic cardiovascular risk factors are associated with preeclampsia: Maternal age >40 years, insulin resistance, obesity, and systemic inflammation and preexisting hypertension, diabetes mellitus, or renal disease all increase the risk.20,25,26 Consistent with this, women with a history of preeclampsia have an elevated risk for cardiovascular disease later in life (see discussion later in this review). Surprisingly, smoking during pregnancy protects against preeclampsia.27The diagnosis of preeclampsia is clinical. As defined by the American College of Obstetrics and Gynecology, the diagnosis requires blood pressures >140/90 mm Hg on 2 occasions combined with urinary protein excretion >300 mg/d. Edema, a classic feature of the disease, is no longer considered a diagnostic feature given its lack of sensitivity or specificity.1 Importantly, in 20% of cases, eclampsia may present without preceding hypertension or proteinuria, suggesting that the currently employed diagnostic criteria are imperfect.28 Laboratory tests, such as liver function tests, quantification of urinary protein, and serum creatinine may be helpful in characterizing the degree of end-organ damage, but none is specific for preeclampsia.1 Hyperuricemia, which is more likely to be present in women with preeclampsia than in normotensive pregnant women, has been used as a diagnostic aid and to predict adverse outcomes in preeclampsia, but its predictive value is generally modest.29,30Recently, work by our group and others has identified an imbalance of proangiogenic and antiangiogenic proteins as a key factor in the pathogenesis of the preeclampsia.31–38 In the present report, we review our current understanding of the biology underlying the disease. We first describe the role of the placenta in preeclampsia, and then review the mechanisms of angiogenesis and its role in preeclampsia and the role of other contributory pathways in the pathogenesis of preeclampsia. Finally, we comment on the potential mechanisms by which the risk of cardiovascular disease is elevated in women with a history of preeclampsia.The Preeclamptic PlacentaThe placenta is the central organ in the pathogenesis of preeclampsia. Removal of the placenta abolishes the disease39; moreover, only the placenta, and not the fetus, is required for its development. This is best demonstrated by the case of molar pregnancy, which carries an elevated risk for preeclampsia.40 Pathological examination of placentas from women with severe preeclampsia reveals several abnormalities including infarcts, atherosis, thrombosis, and chronic inflammation.41 It is likely that some of the abnormalities seen in the preeclamptic placenta are consequences of the hypertension and endothelial injury induced by the disease. However, there are abnormalities of placental development that precede the maternal derangements.During normal placentation, the embryo-derived cytotrophoblast cells invade the maternal uterine wall. After invasion, cytotrophoblasts are found in the smooth muscle and endothelial layers of the maternal decidual arteries. This interaction acts to induce the remodeling of these maternal vessels into high-capacitance and low-resistance vessels that provide access to maternal oxygen and nutrients for the placenta and developing fetus.42 As part of this process, the cytotrophoblasts adopt an endothelial phenotype, expressing adhesion molecules classically found on the surface of endothelial cells.43 In preeclampsia, this process is aberrant. The invasion of the cytotrophoblasts is incomplete, with cytotrophoblast cells present only in the superficial layers of the decidua.43 The spiral arteries fail to be invaded or remodeled, resulting in constricted, high-resistance vessels, visible on pathological examination of preeclamptic placentas. This shallow invasion has been shown to be related to a failure of the cytotrophoblasts to adopt an endothelial adhesion phenotype44 (Figure 1).Download figureDownload PowerPointFigure 1. Abnormal placentation in preeclampsia. In normal placental development, invasive cytotrophoblasts of fetal origin invade the maternal spiral arteries, transforming them from small-caliber resistance vessels to high-caliber capacitance vessels capable of providing placental perfusion adequate to sustain the growing fetus. During the process of vascular invasion, the cytotrophoblasts differentiate from an epithelial phenotype to an endothelial phenotype, a process referred to as pseudovasculogenesis, or vascular mimicry (top). In preeclampsia, cytotrophoblasts fail to adopt an invasive endothelial phenotype. Instead, invasion of the spiral arteries is shallow, and they remain small-caliber resistance vessels (bottom). Figure reproduced with permission from Lam et al.45Hypoxia may contribute to the aforementioned abnormal placental development because the failure of cytotrophoblasts to fully invade and to switch adhesion molecules can also be reproduced in vitro when cytotrophoblasts are cultured under hypoxic conditions.46 Consistent with this, the risk for preeclampsia is higher in women living at high altitude.47 However, hypoxia resulting from abnormal placentation also contributes to the fetal and maternal complications of the disease. Clinically, abnormal uterine artery Doppler waveforms herald the development of preeclampsia, suggesting decreased placental perfusion.48 Decreased placental perfusion in its more extreme cases results in fetal growth restriction, oligohydramnios, or intrauterine fetal demise. Interestingly, pregnant rats and baboons develop hypertension and proteinuria in response to surgically induced uteroplacental ischemia,49,50 implicating placental hypoxia in the development of the maternal disease.Proangiogenic Factors and Vascular HomeostasisAbnormalities in the placenta and resulting consequences to the fetus are a hallmark of preeclampsia, but the maternal features of the disease have been its most mysterious feature. Recently, circulating antiangiogenic proteins have been implicated in the pathogenesis of many of the maternal features of the disease (Figure 2). Before describing the manner in which release of these factors into the circulation may lead to the maternal syndrome, we review the evidence for the role of proangiogenic growth factors and their receptors in vascular homeostasis.Download figureDownload PowerPointFigure 2. sFlt1 and soluble endoglin (sEng) cause endothelial dysfunction by antagonizing vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) signaling. There is mounting evidence that VEGF and TGF-β1 are required to maintain endothelial health in several tissues including the kidney and perhaps the placenta. During normal pregnancy, vascular homeostasis is maintained by physiological levels of VEGF and TGF-β1 signaling in the vasculature. In preeclampsia, excess placental secretion of sFlt1 and sEng (2 endogenous circulating antiangiogenic proteins) inhibits VEGF and TGF-β1 signaling, respectively, in the vasculature. This results in endothelial cell dysfunction, including decreased prostacyclin, nitric oxide production, and release of procoagulant proteins. TβRII indicates transforming growth factor-β type II receptor.Vascular Endothelial Growth FactorsVascular endothelial growth factors (VEGF) are secreted dimeric glycoproteins involved in vasculogenesis (the process by which new blood vessels are formed in embryonic life) and angiogenesis (the process by which blood vessels branch to form new blood vessels). In humans and other mammals, this family of growth factors includes VEGF-A and placental growth factor (PlGF), among others. VEGF-A (hereafter referred to as VEGF), the first discovered and prototypical protein in this family, is a proangiogenic factor that promotes the proliferation and survival of endothelial cells and induces vascular permeability.51,52 PlGF is a VEGF homolog released by the placenta, which also has proangiogenic activity.53Vascular endothelial growth factors family receptors present on vascular endothelial cells include Flt-1 (VEGFR-1) and KDR (VEGFR-2, murine Flk-1). Whereas VEGF binds to both Flt-1 and KDR receptors, PlGF homodimers bind exclusively to Flt-1.54 KDR is thought to be responsible primarily for the action of VEGF on endothelial cells.55–57 KDR-null mice die at embryonic day 8.5 to 9.5 with an absence of organized blood vessels and widespread necrosis, suggesting lack of perfusion to vital structures.58 Flt-1–null mice die in embryonic life, with death due to the overgrowth of endothelial cells and resultant blood vessel disarray.58 Mice with Flt-1 lacking the tyrosine kinase domain but with intact ligand binding domain have normal blood vessels and survive,59 implying that Flt-1 acts as a negative regulator of angiogenesis through sequestration of extracellular VEGF rather than through intracellular action. More recently, work by Chappell et al60 suggests that the role of the Flt-1 gene may be to express sFlt-1 (a soluble VEGF signaling inhibitor), which acts by regulating guidance of emerging vessel sprouts by modulating local VEGF availability.Vascular endothelial growth factors is essential for embryonic vasculogenesis and angiogenesis. The ablation of a single VEGF allele results in markedly abnormal vasculature, including the placental vasculature, with death at embryonic day 10 to 12.61 Besides its essential role in placental and embryonic vasculogenesis and angiogenesis, VEGF is involved in the survival of endothelial cells and vascular homeostasis in mature vessels and tissues. In adult mice, VEGF is expressed by cell types located adjacent to fenestrated endothelia, including the epithelial cells of the choroid plexus, renal podocytes, and hepatocytes.62 In vitro, VEGF induces endothelial fenestrations,63 whereas inhibition of VEGF in adult mice reduces the density of so-called VEGF-dependent fenestrated capillaries.64 Accordingly, targeted inhibition of VEGF in vivo leads to pathology in many of the organs with fenestrated endothelia, which are also affected in preeclampsia. Specifically, in the mouse kidney, podocyte-selective knockout of VEGF in early postnatal life results in proteinuria, nephrotic syndrome, endotheliosis, and eventual disappearance of endothelial cells from the glomerular tuft, recapitulating the classic renal lesion seen in preeclampsia.65 In the liver, inhibition of VEGF signaling in early postnatal life leads to abnormal liver development, with small hepatocytes and immature sinusoidal vasculature.66 In adult mice, activation of the Flt-1 receptor on liver sinusoidal endothelial cells by VEGF or PlGF leads to elaboration of hepatocyte growth factor and liver enlargement.67 Additionally, in the brain, inhibition of VEGF signaling results in decreased perfusion of choroid plexus vasculature.68Vascular endothelial growth factors also seems to have a direct vasodilatory effect on the systemic vasculature because infusion of VEGF leads to nitric oxide–dependent vasorelaxation in the coronary arteries and other vessels in dogs and humans,69 likely through upregulation of nitric oxide and prostacyclin in vascular endothelial cells.70,71 Suggesting a role for VEGF in control of systemic blood pressure, antagonism of the KDR receptor leads to elevations in mean arterial pressure in mice by a nitric oxide–dependent mechanism.72 Most relevant, VEGF inhibition has a biological effect on endothelial function in adult men and women. Side effects of VEGF inhibition in patients undergoing antiangiogenic cancer therapy are consistent with those in animal models, suggesting the homeostatic role of VEGF in the mature vasculature: hypertension, proteinuria, glomerular endothelial damage, hypothyroidism, and, in rare cases, the reversible posterior leukoencephalopathy syndrome.73–78PlGF, which has ≈53% homology with VEGF, is expressed at high levels by the human placenta. PlGF homodimers do not bind to the KDR receptor but bind to the Flt-1 receptor with high affinity. PlGF has weak mitogenic activity and no effect on vascular permeability in vitro alone but potentiates the actions of VEGF in cultured endothelial cells and in an in vivo vascular permeability model.54 In contrast with VEGF knockout mice, PlGF-null mice have normal vascular development with the exception of subtle defects in the retinal vasculature and in luteal vasculogenesis, which do not seem to affect retinal or reproductive functioning.79 However, PlGF-null mice exhibit defects in tumor angiogenesis, postischemic retinal and myocardial neovascularization, and wound healing, suggesting that PlGF plays a role in angiogenesis in pathological settings.79,80 Consistent with this, PlGF stimulates angiogenesis in ischemic myocardium and arterial collateral growth in ischemic limbs. PlGF may act by displacing VEGF from the Flt-1 receptor, allowing it to bind to the more active KDR receptor.54 Other possible mechanisms include direct effects of Flt-1 signaling and the formation of VEGF/PlGF heterodimers.81,82 During pregnancy, the placenta releases PlGF at high amounts into the maternal circulation. Levels increase beginning in the second trimester, peak during weeks 29 to 32, and decline thereafter. However, because most in vivo investigation of PlGF has been conducted in nonpregnant animals, the function of PlGF in the physiology of normal pregnancy has not been well elucidated.Transforming Growth Factor-βThe transforming growth factor-β (TGF-β) family of proteins is made up of ubiquitous growth factors with diverse actions in many cell types. TGF-β is known to be involved in angiogenesis; however, the mechanisms are not as well elucidated as those in the VEGF pathway. To initiate intracellular signaling, TGF-β and other proteins in this family must bind to both type I and type II receptors on the cell surface. TGF-β isoforms bind the TGF-β type II receptor (TGF-βII) initially with subsequent binding and activation of type I receptors. Mice null for the TGF-βII receptor die at embryonic day 10.5 with defects in hematopoiesis and vasculogenesis,83 implicating TGF-β in the development of the vasculature. Most cell types, including endothelial cells, express the activin-like kinase type I TGF-β receptor ALK5, but endothelial cells alone express ALK1 type I receptor.84 Like TGF-β, ALK1 is important in the development of blood vessels because ALK1-null mice perish by embryonic day 11 to 12 with growth retardation with a markedly reduced number of capillaries and dilation of larger vessels.85 In vitro, TGF-β has differing effects on the activation state of endothelial cells dependent on the dose administered: At low TGF-β doses, ALK1 leads to proliferation and migration of endothelial cells, whereas at high TGF-β doses, ALK5 inhibits proliferation and migration of endothelial cells.84,86 Moreover, TGF-β signaling regulates the expression of VEGF, connecting the 2 pathways and further linking TGF-β to angiogenesis.87Transforming growth factor -β signaling in the vasculature also involves coreceptors that act to modulate TGF-β action. Endoglin is a TGF-β coreceptor expressed in endothelial cells and syncytiotrophoblasts of the placenta. Both human endoglin and ALK1 mutations independently cause hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease), characterized by multisystemic vascular malformations.88 Endoglin-null mice die at embryonic day 10 to 12 with abnormal vasculature and internal bleeding.89 On a molecular level, endoglin promotes and is required for TGF-β/ALK1–mediated endothelial cell proliferation and migration.86Besides being involved in angiogenesis in the embryonic phase, TGF-β likely functions in vascular homeostasis in mature vessels. In the mouse brain, TGF-β inhibition in combination with VEGF inhibition resulted in the loss of choroid plexus endothelial fenestrae with formation of periventricular edema detectable on magnetic resonance imaging.68 Downregulation of TGF-β signaling in the retinal microvasculature leads to decreased perfusion, breakdown of the blood-retinal barrier, reduced endothelium-dependent vasodilation, and increased endothelial cell apoptosis, suggesting a role for TGF-β in the maintenance of the adult vasculature.90Antiangiogenic Factors and Endothelial Dysfunction in PreeclampsiaIn 1989, Roberts and Taylor et al91,92 advanced the hypothesis that preeclampsia results from the release of circulating factors by the placenta, leading to widespread maternal vascular endothelial dysfunction. Several lines of evidence continue to support this understanding of the disease. The cardinal signs and symptoms of preeclampsia involve the vasculature, specifically areas of the vasculature with fenestrated endothelia. Furthermore, vessels isolated from the soft tissue of preeclamptic women demonstrate endothelial dysfunction, with impaired endothelium-dependent but not endothelium-independent dilatation.93,94 Human studies have firmly established the presence of factors released by the injured or activated endothelium in the circulation of women with clinical preeclampsia. These include, among others, endothelin-1,95 fibronectin,96–98 von Willebrand factor,96,99 thrombomodulin,100,101 markers of oxidative stress,102 and inflammatory cytokines.103 There is also evidence of deficiency of prostacyclin and nitric oxide, vasodilatory factors released by healthy vasculature, in the circulation of women with preeclampsia.104–106 Studies showing that serum from pregnant women with preeclampsia induces endothelial injury and dysfunction in vitro support the theory that a circulating factor causes the aforementioned endothelial dysfunction evident in the disease.92,102,107sFlt-1 and PlGF in the Pathogenesis of PreeclampsiaBecause the vascular endothelium relies on proangiogenic factors, the release of antiangiogenic factors by the placenta into the maternal circulation is a plausible cause of the endothelial dysfunction observed in preeclampsia. Investigation by our group and others has characterized 2 such antiangiogenic proteins. Soluble fms-like tyrosine kinase (sFlt-1, also referred to as sVEGFR-1), an antiangiogenic protein, is a soluble form of the VEGF/PlGF receptor Flt-1 produced by alternative splicing.108 sFlt-1 was initially identified as a product of cultured human endothelial cells and subsequently shown to be produced by the placenta and released into the maternal circulation.108–110 sFlt-1 is a potent inhibitor of VEGF and PlGF activity: Recombinant sFlt-1 inhibits endothelial tube formation and blocks the vasodilatory effect of VEGF and PlGF in vitro.108 Our group identified elevated expression of sFlt-1 by gene expression profiling in placentas delivered from women with preeclampsia.31 Since that time, several novel sFlt-1 variant isoforms have been identified and shown to be upregulated in preeclampsia.111–113Animal data support a causal role for sFlt-1 in the pathogenesis of the maternal disease. Administration of sFlt-1 to pregnant rats with the use of an adenoviral vector induced hypertension and proteinuria and caused glomerular endotheliosis, the classic renal lesion seen in preeclampsia.31 Other groups have also generated animal models that implicate sFlt-1 in the pathogenesis of preeclampsia. These include rat and baboon models in which uterine hypoxia induced elevated production of sFlt-1, hypertension, and proteinuria,50,114 as well as a mouse model in which sFlt-1 expression in pregnant females resulted in hypertension, decreased platelet count, and reduced fetal weight.115Soluble Endoglin in the Pathogenesis of PreeclampsiaNotably absent from the phenotype of rats administered sFlt-1 are the liver dysfunction and cerebral changes seen in women with severe preeclampsia. Soluble endoglin (sEng) is another antiangiogenic protein identified by gene expression profiling of placentas from women with preeclampsia. sEng may combine with sFlt-1 to induce features of severe preeclampsia including liver dysfunction, fetal growth restriction, coagulation, and neurological abnormalities.32,68 Our group identified the 65-kDa sEng monomer produced by placentas from preeclamptic women at a level 4-fold higher than placentas from women with normal pregnancies. Subsequent in vitro studies demonstrated that sEng reduces the binding of TGF-β1 to its receptor and blocks TGF-β1–induced vasodilation of rat vessels, likely through downregulation of nitric oxide synthase.32 Furthermore, sEng reduced endothelial tube formation in vitro and led to increased capillary permeability in mouse lung, liver, and kidney. Importantly, when pregnant rats were injected with both sFlt-1 and sEng, a condition reminiscent of severe preeclampsia developed with hypertension, nephrotic range proteinuria, low platelet count, elevated liver enyzmes, and reduced fetal weight.32 Thus, most, if not all, clinical manifestations of preeclampsia can be explained by the antiangiogenic actions of sFlt-1 and sEng on the maternal endothelium. More recent studies have shown that mice injected with both sFlt-1 and sEng expressing adenoviruses, but not those injected with adenoviruses expressing either molecule alone, exhibit not only decreased cerebral perfusion and vascular thrombi but also loss of choroid plexus endothelial fenestrae, choroid plexus endothelial swelling, and cerebral edema on brain magnetic resonance imaging.68 Although women with eclampsia are known to have brain edema and white matter lesions on magnetic resonance imaging, it remains to be seen whether the histopathological changes in women with preeclampsia with neurological involvement are similar to those seen in sFlt-1– and sEng-injected mice.Human Studies of sFLT-1, PlGF, and sEng in PreeclampsiaEpidemiological studies have revealed that blood levels of sFlt-1 and PlGF are altered in women with preeclampsia both during and before clinical signs and symptoms of the disease, consistent with a pathogenic role for these angiogenic factors in preeclampsia. sFlt-1 is present at relatively high concentrations in the serum of normal pregnant women at term116 but declines to nonpregnant levels 48 hours after delivery.31 In preeclampsia, sFlt-1 levels begin to rise at least 5 weeks before the onset of clinical disease and remain elevated compared with unaffected women.34,36,117 Alterations in sFlt1 are more dramatic in patients who have early-onset preeclampsia (preeclampsia at <37 weeks).34 Levels of sFlt-1 also correlate with the severity of the disease.37 In pregnancies afflicted by severe intrauterine fetal growth restriction without preeclampsia, there may also be a modest elevation of sFlt-1 levels.118Consistent with the pathophysiology suggested by animal models, levels of free PlGF are depressed in women with preeclampsia. In fact, low PlGF levels in the first trimester, before the sFlt-1 rise, are a risk factor for subsequent preeclampsia.34,119 PlGF can also be measured in the urine of women destined to develop preeclampsia, where levels are depressed compared with normotensive pregnant women beginning at 25 weeks. The degree of suppression of urinary PlGF levels is correlated with the severity of the disease.120 In contrast, although circulating free VEGF levels are low in preeclampsia, it is not useful clinically because the majority of patients have levels below the detection limit of the currently available enzyme-linked immunosorbent assay kits. Of note, the ratio of sFlt-1 to PlGF is a better marker of preeclampsia than either measure alone.121,122 This implies that an imbalance of antiangiogenic and proangiogenic factors rather than the level of either sFlt-1 or PlGF alone leads to preeclampsia.34Studies of sEng levels in women with and without preeclampsia are consistent with the animal studies, supporting a role for elevated sEng in the pathogenesis of severe preeclampsia. sEng levels in women with normal pregna