• E-mail
• Print
• Comment
• Font Size
• Digg
• del.icio.us
• Discuss article

Plasma Soluble Vascular Endothelial Growth Factor Receptor-1 Concentration is Elevated Prior to the Clinical Diagnosis of Pre- Eclampsia

Posted on: Sunday, 24 April 2005, 03:00 CDT

Abstract

Objective: Accumulating evidence suggests that the balance between vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and their receptors is important for effective vasculogenesis, angiogenesis, and placental development. Recently, the soluble form of VEGFR-1 (sVEGFR-1), an antagonist to VEGF and PlGF, has been implicated in the pathophysiology of pre-eclampsia. Plasma sVEGFR-1 concentration is elevated in pre-eclampsia at the time of clinical diagnosis and correlates with the severity of the disease. The purpose of this study was to determine whether the concentrations of sVEGFR-1 in plasma of pre-eclamptic patients change prior to the clinical manifestations of the disease.

Methods: A longitudinal case-control study was conducted in normal pregnant women (n=44) and patients with preeclampsia (n=44). Blood sampling was performed at six intervals: (1) 7-16 weeks; (2) 16-24 weeks; (3) 24-28 weeks; (4) 28-32 weeks; (5) 32-36 weeks; and (6) more than 37 weeks of gestation. To examine the relationship between plasma sVEGFR-1 concentration and interval to clinical diagnosis of pre-eclampsia, plasma samples of pre-eclamptic patients at different gestational ages were stratified according to the interval from blood sampling to clinical development of the disease into five groups: (1) at clinical manifestation; (2) 2-5 weeks; (3) 6-10 weeks; (4) 11-16 weeks; and (5) 17-25 weeks before clinical manifestations. Plasma concentrations of sVEGFR-1 were determined by enzyme-linked immunoassay. Parametric statistics and repeated measure procedures were used for the analysis.

Results: The mean plasma sVEGFR-1 concentration in pre-eclamptic patients before the clinical manifestation of the disease was significantly higher than in normal pregnant women at 24-28, 28-32, and 32-37 weeks of gestation (p=0.02, p < 0.001, and p < 0.001, respectively). In contrast, no significant differences in the mean plasma sVEGFR-1 concentration between patients with pre-eclampsia and normal pregnant women were observed both at 7-16 weeks and 16- 24 weeks of gestation (p=0.1 and p=0.9). Similarly, the mean plasma sVEGFR-1 concentration was significantly higher in preeclamptic patients than in normal pregnant women at clinical manifestation, at 2-5 weeks (mean 3.8 weeks), and at 6-10 weeks (mean 8.2 weeks) prior to the development of clinical pre-eclampsia (p < 0.001, p < 0.001, and p=0.002, respectively). Among patients with early-onset pre- eclampsia (defined as gestational age of 34 weeks or less), the mean plasma sVEGFR-1 concentration was significantly higher in pre- eclampsia (before clinical diagnosis) than in normal pregnant women at 24-28 (mean 26.4) weeks of gestation (p=0.008). In contrast, among patients with the late-onset disease (defined as gestational age of more than 34 weeks), plasma sVEGFR-1 concentration in pre- clinical pre-eclampsia was significantly higher than in normal pregnant women at 28-32 (mean 30.2) weeks of gestation (p < 0.001).

Conclusions: Plasma sVEGFR-1 concentration is elevated in pre- eclampsia prior to the clinical diagnosis of the disease. This elevation began 6-10 weeks prior to the clinical manifestations, and the increase was more pronounced at 2-5 weeks before the diagnosis, as well as at clinical presentation. Furthermore, in early-onset pre- eclampsia, plasma concentration of sVEGFR-1 is elevated earlier than the late-onset disease.

Keywords: Pre-eclampsia, plasma-soluble vascular endothelial growth factor 1, longitudinal study, early onset, late onset, VEGF, PlGF

Introduction

Pre-eclampsia, a syndrome diagnosed by the presence of hypertension and proteinuria [1-10], has been proposed to be a two- stage disease [4] in which abnormal placentation [8,11-14] is followed by generalized endothelial cell dysfunction [15]. For decades, the term "toxemia of pregnancy" was used to refer to the disease, implying that an unknown "toxic factor(s)" produced by the placenta gains access into maternal circulation, causing systemic endothelial dysfunction, hypertension, and multi-organ involvement/ damage [16-24].

Two angiogenic factors, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), are important for effective function of endothelial cells and placental development [25-30]. VEGF can promote endothelial cell proliferation, migration [31], and survival [32], and exerts its biologic effects through two high- affinity tyrosine kinase receptors: VEGFR-I (VEGF receptor-1 or flt- 1 or fms-like tyrosine kinase-1) and VEGFR-2 (VEGF receptor-2 or KDR/ Flk-1 or kinase domain receptor). VEGFR-I has two isoforms: a transmembranous form and a soluble form (sVEGFR-1). The latter is generated by a splice variant of the VEGFR-I gene and contains the extracellular ligand-binding domain, while lacking the signaling tyrosine kinase domain. Thus, this isoform binds VEGF and inhibits its biological activities [31]. Another ligand for VEGFR-I is PlGF, whose function is to potentiate the angiogenic effects of VEGF. Whereas VEGFR-2 is the major mediator of the mitogenic, angiogenic, permeability-enhancing, and endothelial survival effects of VEGF, the precise function of VEGFR-1 is still a subject of debate [31]. Most investigators believe that VEGFR-1 may not primarily be a receptor transmitting a mitogenic signal, but rather a "decoy" receptor able to inhibit the activity of VEGF on vascular endothelium by preventing binding of VEGF to VEGFR-2. However, there is evidence suggesting that VEGFR-1 signaling can be functional, especially in pathologic conditions such as ischemic limb, retina, or heart [33-35].

A role for the balance between angiogenic/anti-angiogenic factors in the genesis of pre-eclampsia has been proposed by several investigators [25,27-30,36-56]. However, a major set of recent observations have been made by the group of Maynard and Karumanchi [57], who proposed that a blockage of VEGF plays a major role in the genesis of the disease. Evidence in support of this includes: (1) the placenta of patients with pre-eclampsia over-expresses s-VEGFR- 1 mRNA and protein [55,57]; (2) the median plasma/serum concentration of sVEGFR-1 is higher in pre-eclampsia at the time of diagnosis than in patients with normal pregnancies [55,57,58] and the plasma concentration correlates with the severity of the disease [59]; (3) pre-eclampsia is associated with decreased plasma/serum concentrations of VEGF and PlGF [47,53,57,6O]; (4) serum of pregnant women with pre-eclampsia have anti-angiogenic effects in the endothelial cell tube formation bioassay and these effects can be restored by the addition of VEGF and PlGF to serum [57]; and (5) administration of sVEGFR-1 to pregnant animals can induce the clinical manifestations of preeclampsia, including hypertension and proteinuria [57]. Moreover, these animals develop glomerular endotheliosis (a pathologic change observed) [61-63], but not pathognomonic of pre-eclampsia [64].

A critical question is, however, whether the increase in plasma sVEGFR-1 occurs before or only at the time of the diagnosis of the disease. The purpose of this study was to determine whether the plasma concentrations of the sVEGFR-1 increase prior to the diagnosis of pre-eclampsia.

Patients and methods

Study design

A retrospective longitudinal case-control study was conducted by searching our clinical database and bank of biologic samples. This study included patients with pre-eclampsia (n = 44) and normal pregnant women (n = 44). All women were enrolled in the prenatal clinic at the Sotero del Rio Hospital, Santiago, Chile, and followed until delivery. Prenatal visits were scheduled at 4-week intervals in the first and second trimester, and every two weeks in the third trimester until delivery. Blood sampling was performed at enrollment and during every visit, whenever the patient consented. Pre- eclampsia was defined as hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg on at least two occasions, 4 h to 1 week apart) and proteinuria (≥ 300 mg in a 24-h urine collection or one dipstick measurement ≥ 2 +) [65]. Severe pre-eclampsia was defined as either severe hypertension (diastolic blood pressure ≥ 110 mmHg) plus mild proteinuria or mild hypertension plus severe proteinuria (a 24-h urine sample containing 3.5 g protein or urine specimen ≥ 3 + protein by dipstick measurement) [65]. Patients with an abnormal liver function test (aspartate aminotransferase > 70 IU/L) plus thrombocytopenia (platelet count < 100,000/cm^sup 3^), as well as those with eclampsia, were also classified as having severe pre- eclampsia [65]. Pregnant women were considered normal if they had no medical, obstetrical or surgical complications, and delivered a normal term (≥ 37 weeks) infant whose birthweight was appropriate for gestational age (10th-90th percentile). The exclusion criteria comprised: (1) known major fetal anomaly or fetal demise; (2) active vaginal bleeding; (3) multifetal pregnancy; (4) serious medical illness (renal insufficiency, congestive heart disease, chronic respiratory insufficiency, etc.); (5) chronic hypertension, asthma requiring medication; and (6) patients requiring anti-platelet or non-steroidal anti\-inflammatory drugs. For this study, subjects were selected only if they had plasma samples available at least once before 24 weeks of gestation, and at least another sample after this gestational age with a total of at least three samples during pregnancy. Plasma samples were selected from each patient only once for each of the following six intervals: (1) 7-16 weeks; (2) 16-24 weeks; (3) 24-28 weeks; (4) 28-32 weeks; (5) 32-37 weeks; and (6) more than 37 weeks of gestation. For each pre-eclamptic case, one control was selected by matching for gestational age ( 2 weeks) at the time of clinical diagnosis of pre- eclampsia. Since the clinical presentation of pre-eclampsia developed at different gestational ages and all control cases delivered at term, more plasma samples from normal pregnant women were included in the study. In an effort to reduce the unequal number of plasma samples between cases and controls, samples from normal pregnant women obtained at gestational ages beyond the time the diagnosis of pre-eclampsia was made in the matched case were not used for this study. The collection and utilization of the samples was approved by both the Human Investigation Committee of the Sotero del Rio Hospital, Santiago, Chile (a major affiliate of the Catholic University of Santiago) and the IRB of the National Institute of Child Health and Human Development. Many of these samples were used in previous studies.

Sample collection and human sVEGFR-1 immunoassay

Venipuncture was performed and blood collected into tubes containing EDTA. The samples were centrifuged and stored at -70C. The concentrations of sVEGFR-1 were measured using an enzyme-linked immunoassay (ELISA; R&D Systems, Minneapolis, MN). This assay employs the quantitative sandwich immunoassay technique. Briefly, recombinant human VEGFR-1 standards and maternal plasma specimens were incubated in duplicate wells of the microtiter plates pre- coated with monoclonal antibodies specific for VEGFR-1. During this incubation, the immobilized antibodies in the microtiter plate bound the VEGFR-1 in both the standards and samples. After washing unbound substances, polyclonal antibodies to human VEGFR-1 conjugated to an enzyme (horseradish peroxidase) were added to the assay wells. After an incubation period, the assay plates were washed to remove unbound antibody-enzyme reagents. Upon addition of a substrate solution (tetramethylbenzidine), color developed in the assay plates proportionally to the amount of VEGFR-1 bound in the initial step. The microtiter plates were read with a programmable spectrophotometer (Ceres 900 Microplate Workstation, Bio-Tek Instruments, Winooski, VT). The inter- and intra-assay coefficients of variation (CVs) were 4.8% and 6.9%, respectively. The sensitivity was 17.8 pg/ml.

Statistical analysis

Kolmogorov-Smirnov tests were used to test for normal distribution of the data. After logarithmic transformation (Log sVEGFR-1), unpaired Student's r-tests or analysis of variance (ANOVA) and post hoc tests with Bonferroni or Dunnett's T3 correction for multiple comparisons were utilized to determine the differences of the mean between and among groups, respectively. Analysis of covariance (ANCOVA) was used to assess the difference in plasma concentrations of sVEGFR-1 between patients destined to develop pre-eclampsia and in normal pregnancy after adjusting for gestational age at blood sampling and intervals of sample storage. Chi-square or Fisher's Exact tests were employed for comparisons of proportions. Repeated-measure analysis was used to examine the difference of the changes in plasma sVEGFR-1 concentrations in relationship to various intervals of blood sampling between normal pregnant women and patients with pre-eclampsia. The statistics package used was SPSS V. 12 (SPSS Inc., Chicago, IL). Significance was assumed for a p value of less than 0.05.

Results

Clinical characteristics of the study population are displayed in Table I. Patients with pre-eclampsia were slightly younger than normal pregnant women and had a history of smoking less frequently than patients in the control group. The group with pre-eclampsia included more nulliparous women than the control group. As expected, patients with pre-eclampsia delivered earlier and had a lower mean birthweight than normal pregnant women. Sixteen (36%) patients with pre-eclampsia delivered neonates whose birthweights were less than the 10th percentile for gestational age. The clinical characteristics of patients with pre-eclampsia are displayed in Table II. Thirty-two (72%) patients had severe pre-eclampsia, while ten (22%) had early-onset pre-eclampsia (defined as gestational age at 34 weeks or less).

Table I. Clinical characteristics of the study population.

Table II. Clinical characteristics of patients with pre- eclampsia.

Table III. Plasma sVEGFR-1 concentrations in normal pregnancy and pre-eclampsia.

The gestational age at which pre-eclampsia was diagnosed varied. Four patients had hypertension and proteinuria at 28-32 weeks, 13 at 32-37 weeks, and 27 at term (≥ 37 weeks). There were no significant differences in the mean gestational age at which venipuncture was performed by the interval window (for sVEGFR-1 plasma determination, see above) between the group of patients who eventually developed pre-eclampsia and the control group. No significant differences in the mean plasma sVEGFR-1 concentration between patients with pre-eclampsia and normal pregnant women were observed both at 7-16 weeks and 16-24 weeks of gestation (p = 0.1 and p = 0.9; see Table III and Figure 1a,b). However, at 24-28, 28- 32, and 32-37 weeks of gestation, the mean plasma sVEGFR-1 concentrations in women who subsequently developed pre-eclampsia were higher than in normal pregnant women (p = 0.02, p < 0.001 and p < 0.001, respectively; see Table III and Figure 1c,d,f). Patients with pre-eclampsia at the time of clinical diagnosis had a significantly higher mean plasma sVEGFR-1 concentration than those before clinical manifestations at 32-37 weeks of gestation (p < 0.001; see Figure 1e) and higher than normal pregnant women both at 32-37 weeks and at term (p < 0.001 for both comparisons; see Figure 1e, f).

Figure 1. Mean plasma sVEGFR-1 concentration of normal pregnant women and patients with pre-eclampsia according to the interval of gestational age at blood samplings. No significant differences in the mean plasma sVEGFR-1 concentration between patients with pre- eclampsia and normal pregnant women were observed both at 7-16 weeks (a) and 16-24 weeks of gestation (b) (p = 0.1 and p = 0.9; see Table III). However, at 24-28 (c), 28-32 (d) and 32-37 weeks of gestation (e), the mean plasma sVEGFR-1 concentrations in pre-eclamptic patients before the clinical manifestations of the disease were higher than in normal pregnant women (p = 0.02, p < 0.001 and p < 0.001, respectively; see Table III). Pre-eclamptic patients at the time of clinical diagnosis had a mean plasma sVEGFR-1 concentration significantly higher than those before clinical manifestations at 32- 37 weeks of gestation (e; p < 0.001), and higher than normal pregnant women both at 32-37 weeks and at term (p < 0.001 for both comparisons; e and f). The statistical tests used were unpaired Student's t-test or ANOVA with Bonferroni correction for multiple comparisons. The vertical axis in the figure is in the logarithmic scale.

To examine the relationship between plasma sVEGFR-1 concentration and the interval to clinical diagnosis of pre-eclampsia, plasma samples of pre-eclamptic patients at different gestational ages were stratified according to the interval from blood sampling to clinical diagnosis into five groups: (1) at clinical diagnosis; (2) 2-5 weeks before diagnosis; (3) 6-10 weeks before diagnosis; (4) 11-16 weeks before diagnosis; and (5) 17-25 weeks before diagnosis. The plasma samples from normal pregnant women (who were matched with patients who developed pre-eclampsia; see Patients and methods) were matched for gestational age with the plasma samples of pre-eclamptic patients at different gestational ages according to the intervals pre-specified above (e.g. 2-5 weeks before diagnosis, etc.). The mean plasma sVEGFR-1 concentration was significantly higher in patients who developed pre-eclampsia than in normal pregnant women at clinical diagnosis, at 2-5 weeks (mean 3.8 weeks), and at 6-10 weeks (mean 8.2 weeks) before the clinical diagnosis (p < 0.001, p < 0.001 and p = 0.002, respectively; see Table IV and Figure 2a-c). The results remained significantly different even after adjusting for gestational age at blood sampling and intervals of sample storage (all p < 0.05). No significant differences in the mean plasma sVEGFR-1 concentration were observed between patients who eventually developed pre-eclampsia and the normal control group, both between 11 and 16 weeks before the diagnosis and between 17 and 25 weeks before clinical manifestations of pre-eclampsia (p = 0.6 and p = 0.5, respectively, see Table IV and Figure 2d,e).

Table IV. Plasma sVEGFR-1 concentrations in normal pregnant women and pre-eclampsia.

Since clinical pre-eclampsia developed at various gestational ages, performing repeated-measure analysis with samples stratified by gestational age alone would make the results difficult to interpret. Therefore, repeated measure analysis was conducted including samples from the following intervals: (1) 17-25 weeks before the diagnosis - the mean interval from blood sampling to the clinical diagnosis was 20.6 weeks and the mean gestational age at sampling was 16.5 weeks; (2) 11-16 weeks before the diagnosis - the mean interval from blood sampling to clinical diagnosis was 13.2 weeks and the mean gestational age at sampling was 24.2 weeks; (3) 6- 10 weeks before the diagnosis - the mean interval from blood sampling to clinical diagnosis of 8.2 weeks and mean gestational age at diagnosis was 28.5 weeks; \(4) 2-5 weeks before the clinical diagnosis - the mean interval from blood sampling to the clinical diagnosis was 3.8 weeks and the mean gestational age was 32.8 weeks; and (5) at clinical diagnosis of pre-eclampsia. Only those patients who donated a blood sample in these intervals were included in the repeated measure analysis.

Figure 2. Mean plasma sVEGFR-1 concentration of normal pregnant women and patients with pre-eclampsia stratified according to the interval from blood sampling to clinical diagnosis of pre- eclampsia. The mean plasma sVEGFR-1 concentration was significantly higher in patients who developed pre-eclampsia than in normal pregnant women at clinical diagnosis (a), at 2-5 weeks (b), and at 6- 10 weeks (c) before the clinical diagnosis (p < 0.001, p < 0.001 and p = 0.002, respectively; see Table IV). No significant differences in the mean plasma sVEGFR-1 concentration were observed between patients who eventually developed pre-eclampsia and the normal control group both between 11 and 16 weeks (e) before the diagnosis and between 17 and 25 weeks (f) before clinical manifestations of pre-eclampsia (p = 0.6 and p = 0.5, respectively, see Table IV). The statistical test used was unpaired Student's t-test. The vertical axis in the figure is in the logarithmic scale.

The results demonstrated that patients who subsequently developed pre-eclampsia (n = 10) had a significantly different profile (plasma concentration over time) of plasma sVEGFR-1 concentration than patients with normal pregnancies (n =10) as a function of the interval of blood sampling to the diagnosis of the disease (p = 0.01; repeated measure analysis; see Figure 3). The mean plasma sVEGFR-1 concentration was higher in pre-eclamptic patients than in normal pregnant women at 6-10 weeks before clinical manifestations, and the increase was more pronounced at 2-5 weeks before as well as at the time pre-eclampsia was diagnosed, but not before 11-25 weeks prior to clinical manifestations ( see Figure 3).

Figure 3. Mean plasma sVEGFR-1 concentration of normal pregnant women and patients with pre-eclampsia stratified according to the interval from blood sampling to clinical diagnosis of pre-eclampsia derived from repeated measure analysis. Patients who subsequently developed pre-eclampsia (n = 10) had a significantly different profile of plasma sVEGFR-1 concentration than patients with normal pregnancies (n = 10) as a function of the interval of blood sampling to the diagnosis of the disease (p = 0.01). The mean plasma sVEGFR- 1 concentration in pre-eclamptic patients was higher than normal pregnant women at 6-10 weeks before clinical manifestation, and the increase was more pronounced at 2-5 weeks before as well as when pre- eclampsia was diagnosed, but not before (11-25 weeks prior to clinical manifestations). The analysis was performed after logarithmic transformation.

Figure 4. Mean plasma sVEGFR-1 concentration of normal pregnant women stratified according to the interval of gestational age at blood samplings derived from repeated measure analysis. The mean plasma sVEGFR-1 concentration in normal pregnancy (n = 19) increased with advancing gestational age with a small increase at 24-28 weeks (1st interval vs 3rd interval; p = 0.035) and a higher rise after 28- 32 weeks interval (4th interval vs 5th interval; p = 0.001, repeated measure analysis). The analysis was performed after logarithmic transformation.

To examine the relationship between the changes of plasma sVEGFR- 1 concentration as a function of gestational age in normal pregnant women, those patients who donated a blood sample in the six following intervals were included in repeated measure analysis (n = 19): (1) 7-16 weeks; (2) 16-24 weeks; (3) 24-28 weeks; (4) 28-32 weeks; (5) 32-37 weeks; and (6) more than 37 weeks of gestation. In normal pregnant women, the mean plasma sVEGFR-1 concentration increased with advancing gestational age with a small increase at 24- 28 weeks (1st interval vs 3rd interval; p = 0.035) and a higher rise after 28-32 weeks interval (4th interval vs 5th interval; p = 0.001; repeated-measure analysis; see Figure 4).

To examine the diagnostic potential of plasma sVEGFR-1 concentrations to identify those destined to develop pre-eclampsia, patients were stratified into early-onset pre-eclampsia (defined as gestational age of 34 weeks or less) and late-onset pre-eclampsia (defined as gestational age of more than 34 weeks). The gestational ages at which the plasma sVEGFR-1 concentration in pre-eclampsia began to rise above those of normal pregnant women varied. For patients with early-onset pre-eclampsia, the mean plasma sVEGFR-1 concentration was significantly higher in pre-eclampsia (before clinical diagnosis) than in normal pregnancy starting around 24-28 (mean 26.8) weeks of gestation (p = 0.008; see Table V). In contrast, for patients with late-onset pre-eclampsia, the plasma sVEGFR-1 concentration in pre-clinical pre-eclampsia was significantly higher than in normal pregnancy at 28-32 (mean 30.3) weeks of gestation (p < 0.001; see Table VI).

Table V. Plasma sVEGFR-1 concentrations in normal pregnant women and patients who developed clinical pre-eclampsia at 34 weeks of gestation or less.

Using a cut-off of 1560 pg/ml (which represents 2 SD above the mean for pregnant women at the same gestational age range) for gestational age 24-28 weeks, a high plasma sVEGFR-1 concentration had a sensitivity and specificity of 16.7% (1/6) and 97.4% (37/38), respectively, to identify early-onset pre-eclampsia (at 29-34 weeks). In contrast, using the cut-off of 1575 pg/ml for gestational ages of 28-32 weeks, the sensitivity and specificity were 83% (5/6) and 95% (40/42), respectively, to identify early-onset pre- eclampsia (at 32-34 weeks).

Similarly, using the cut-off of 1575 pg/ml for gestational ages of 28-32 weeks, high plasma sVEGFR-1 concentration had a sensitivity and specificity of 18.5% (5/27) and 95% (40/42), respectively, for the identification of late-onset pre-eclampsia (at more than 34 weeks). In contrast, using the cut-off of 2164 pg/ml for gestational ages ranging from 32-36 weeks (mean 34 weeks), the sensitivity and specificity were 70% (14/20) and 97% (36/37), respectively, to predict late-onset pre-eclampsia (at 37 weeks or more).

Discussion

Our results demonstrate that: 1) women destined to develop pre- eclampsia have a higher plasma sVEGFR-1 concentration than those who will have a normal pregnancy; and 2) the increase is detectable several weeks prior to the clinical recognition of the disease. Such change could be detected about 6-10 weeks prior to the clinical diagnosis, and was more pronounced 2-5 weeks before diagnosis and at the time of clinical presentation. Moreover, both early-onset (≤ 34 weeks) and late-onset (> 34 weeks) pre-eclampsia were associated with an elevation of plasma sVEGFR-1 concentration prior to the diagnosis.

The observation that plasma sVEGFR-1 concentration in pre- eclamptic patients was elevated prior to the clinical diagnosis is consistent with the study of Levine et al., who reported that serum concentration of sVEGFR-1 increased approximately five weeks , before the onset of the disorder [66]. Indeed, the view that pre- eclampsia is a chronic process with a long sub-clinical phase was first recognized by Gant et al. in 1974, who demonstrated that patients destined to develop pre-eclampsia had an abnormal angiotensin II sensitivity test as early as the 22nd week of gestation [67,68]. Similarly, abnormal uterine artery Doppler velocimetry [69] (defined as the mean resistance index above 95th percentile for gestational age [70] or the presence of bilateral notching [71]), an index of the increased impedance to blood flow in the uterine artery [72-77], is present weeks before the onset of clinical hypertension and proteinuria in a subset of patients with pre-eclampsia [78-86].

Table VI. Plasma sVEGFR-1 concentrations in normal pregnant women and patients who developed clinical pre-eclampsia at more than 34 weeks of gestation.

The elevation of plasma sVEGFR-1 in early-onset pre-eclampsia was first observed a few weeks (24-28 weeks) after the reported persistence of abnormal uterine artery Doppler in pre-eclamptic patients, suggesting the existence of a temporal relationship between failure to increase utero-placental perfusion and elevated plasma sVEGFR-1 concentration. It is possible that inadequate perfusion on the growth of feto-placental unit may lead to an increase in plasma sVEGFR-1 concentration. Consistent with this hypothesis is that plasma sVEGFR-1 concentration in pre-eclampsia at the time of clinical diagnosis is associated with the abnormality of uterine artery Doppler velocimetry, and that there is an inverse relationship between plasma sVEGFR-1 concentration and birthweight, as well as adjusted birthweight for gestational age (multiple of median), suggesting that an elevated plasma sVEGFR-1 concentration is indicative of a problem with the utero-placental supply line (Chaiworapongsa et al., to be submitted). The findings that plasma sVEGFR-1 concentration is elevated both in early-onset and late- onset pre-eclampsia suggest that these two conditions have a common final pathophysiological pathway. Perhaps an anti-angiogenic state is part of the common pathway of pre-eclampsia.

Several lines of evidence indicate that the source of the plasma sVEGFR-1 is likely to be the placenta. Immunohistochemistry and in situ hybridization studies localized VEGFR-1 protein and mRNA to extravillous trophoblast and villous cytotrophoblast, and more intense staining has been observed in syncytiotrophoblast in the latter half of gestation [87-89]. Clark et al. demonstrated that both membranous and soluble forms of VEGFR-1 were detectable in the placenta, and that sVEGFR-1 protein could be found in the supernatant from explants of placental villi [38], suggesting that sVEGFR-1 could be releas\ed into the intervillous space. Indeed, the sVEGFR-1 concentrations obtained from cytotrophoblast-conditioned medium were higher in tissues derived from pre-eclamptic patients than from those of normal pregnancies [25]. The expressions of both membranous and soluble forms of VEGFR-1 mRNA and protein are also up- regulated in the placenta of pre-eclamptic patients [57].

The mechanisms responsible for the elevation of plasma sVEGFR-1 concentration in patients with pre-eclampsia remain to be determined. Since experimental evidence suggests that hypoxia could stimulate VEGFR-1 expression in trophoblast [90-92], it is possible that an ischemic placenta could induce expression and release sVEGFR- 1 from villous trophoblast, which is in direct contact with maternal blood in the intervillous space. Evidence in support of this includes: (1) increased expression of VEGFR-1 protein in villous trophoblast of patients with pre-eclampsia and some patients with SGA [93]; (2) increased mRNA expression for both membrane and soluble forms of VEGFR-1 in villous trophoblast of placenta obtained from patients with ischemic villi [94]; and (3) increased expression of VEGFR-1 mRNA in the placenta of pre-eclamptic patients, but not from the placenta of neonates born with severe hypoxic ischemic encephalopathy has been reported [95]. This evidence also suggests that a chronic process, rather than acute severe hypoxia, is associated with an up-regulation of VEGFR-1 expression.

Alternatively, since the function of VEGFR-1 and its ligands (VEGF and P1GF) are involved in angiogenesis and trophoblast proliferation/differentiation [36,39,44,96,97], it is possible that the increased expression of VEGFR-1 in trophoblast is associated with the structural remodeling process of the villous tree and blood vessels [98-102] in response to chronic depletion of utero- placental nutrients (e.g., oxygen, but also amino acid, glucose, etc.). Indeed, previous morphological studies of the villous tree indicate that pre-eclampsia is associated with excessive branching capillary network (branching angiogenesis) in peripheral placental villi [98,99], a process thought to be an adaptive response of the fetus to increase gas-nutrient exchange [103]. Moreover, plasma concentrations of sVEGFR-1 in patients with pre-eclampsia and those with SGA with abnormal uterine artery Doppler are related inversely to gestational age at clinical diagnosis (Chaiworapongsa et al., to be submitted), probably due to the need for more extensive remodeling of the villous tree in earlier gestation. Furthermore, our previous observations in patients with pre-eclampsia and those with SGA with abnormal uterine artery Doppler at the time of clinical diagnosis indicate that the increased impedance to blood flow in the villous circulation as measured by abnormal umbilical artery Doppler velocimetry is associated with an increased plasma sVEGFR-1 concentration (delta value). There is evidence suggesting that impedance to flow in the umbilical arteries is a function of the number of tertiary stem villi and their morphology [100,104- 106].

Why does trophoblast release s VEGFR-1 during utero-placental insufficiency? In mammals, growth of the fetus is exponential during the last half of gestation, whereas growth of the placenta is slower [48]. Unless the efficiency of placental transport of nutrients is increasing to keep pace with the demand of the fetus, fetal growth could be compromised. Placental transport capacity could increase as gestational age advances by two mechanisms [48]: (1) increased uterine and umbilical blood flow, and (2) increased rate of nutrient extraction. Evidence from animal experiments suggests that the former seems to be a preferable method [107] and, thus, failure to increase utero-placental perfusion during mid-gestation could compromise fetal growth. Villous "maturation" and "differentiation" could be considered as established responses to increase the rate of nutrient extraction [98,103]. The normal villous maturation involves differential growth of capillaries by endothelial cell proliferation followed by remodeling, as well as morphological changes from stem villi and immature intermediate villi (branching angiogenesis) to mature intermediate villi and terminal villi (non-branching angiogenesis) [98,103]. This mechanism would be more important in cases of chronic limitations to the utero-placental blood flow (e.g., pre-eclampsia). The period between mid-gestation and the start of the third trimester is an important transitional phase in villous development (angiogenic switch). The growth of capillaries rises dramatically at 20-24 weeks of gestation, and remodeling or enlargement occurs diereafter. Similarly, the preferential growth of terminal villi (the most efficient villi for nutrient exchange) occurs by 26 weeks of gestation [98,101], Our observation that plasma sVEGFR-1 concentration of pre-eclamptic patients began to rise at 24-28 weeks in early-onset pre-eclampsia and at 28-32 weeks for the late-onset disease suggests that a problem in the supply line (e.g., inadequate utero-placental perfusion) may occur at different stages of angiogenesis and structural development of the villi. This hypothesis could explain, at least in part, the various villous morphologic findings between the early-onset and the late- onset disease. Early-onset disease is associated with poorly developed and non-branching villous capillaries, while late-onset pre-eclampsia is associated with abundant villous branching [27,98,99,101,106]. As a result, the prevalence of abnormal umbilical artery Doppler is higher in early-onset than that in the late-onset disease [108-110].

VEGFR-1 has been localized to villous trophoblast and endothelial cells of villous blood vessels, suggesting that this protein may play an autocrine role in trophoblast function and a paracrine role in vascular growth at feto-maternal interface. Clark et al. observed that the expression of VEGFR-1 mRNA in villous trophoblast varied considerably even in a single small villus [38,89]. This indicated that VEGFR-1 production is regulated in a specific manner and may play an important role in villus development. However, the precise function of the membrane and the soluble form of VEGFR-1 in the development of human villous tree remains unclear.

Roberts and Lain proposed that the pathophysiology of pre- eclampsia consists of two stages: utero-placental insufficiency, followed by generalized endothelial cell dysfunction [4]. The soluble form of VEGFR-1 could be one of several factors linking these two pathological processes in pre-eclampsia.

The clinical consequence of elevated plasma sVEGFR-1 concentration could be the alteration of endothelial cell function. Normal nonpregnant women have low levels of serum VEGF [46], which is thought to be required for the maintenance of endothelial cell function and survival [32,111,112]. The high plasma sVEGFR-1 concentration observed herein may interfere with this physiological process. Plasma VEGF and P1GF, two important proangiogenic factors, increased during normal pregnancy, but decreased in pre-eclampsia weeks before the clinical diagnosis [51,52,113-115]. Since sVEGFR-1 binds to VEGF and P1GF with a higher affinity than VEGFR-2 [31,116], a functional receptor of endothelium, the increased plasma sVEGFR-1 observed in pre-eclampsia would result in a net effect of anti- angiogenesis [57].

Moreover, the increased availability of sVEGFR-1 in pre- eclampsia may counteract the nitric oxide [117-120]/prostacyclin- induced [120,121] vasodilatation effect of VEGF and result in the elevation of maternal blood pressure. Consistent with this hypothesis, several recent studies in both animals and humans have suggested a role for the blockade of VEGF action that could induce clinical hypertension and proteinuria [57,122,123,124]. However, the effect of chronic elevation of sVEGFR-1 to human endothelial cells during pregnancy requires further investigation.

It is likely that factors other than sVEGFR-1 participate in the pathophysiology of pre-eclampsia [3,4]. The alterations of endothelial cell function depend not only on anti-angiogenic factors, but on others as well. Examples of factors detrimental to endothelial cells include lipid peroxides [125-128] and oxidative stress [129-132], while examples of protective factors are growth factors [32,111] and anti-oxidant agents [133-135]. The balance between the detrimental and protective factors of endothelial cells could determine the susceptibility of the patients and clinical presentation; for example, whether the patients will develop hypertension and proteinuria or not. An extensive longitudinal study [136] of biochemical markers in serum of pre-eclamptic patients indicated that the concentration of triglyceride is higher, but high- density lipoprotein cholesterol, ascorbic acid, and PIGF are lower in serum of patients destined to develop pre-eclampsia compared to those in normal pregnant women at 20 weeks of gestation. This finding may indicate that pre-eclamptic patients are more susceptible to generalized endothelial cell dysfunction beginning in early pregnancy. The increased plasma sVEGFR-1 concentration in the early third trimester would be a second-hit phenomenon. Consistent with this hypothesis is that non-pregnant women with a history of pre-eclampsia had higher plasma concentrations of low-density lipoprotein cholesterol and triglyceride and a higher susceptibility to lipoprotein oxidation when compared with women who had a normal pregnancy at 1-3 years after delivery [137]. Similarly, non- pregnant women who had a history of pre-eclampsia had a significantly decreased total anti-oxidant status than those who never had the condition [138].

While examining the diagnostic potential for plasma sVEGFR-1 concentration, we used the cut-off derived from plasma sVEGFR-1 concentration which is two standard de\viations above the mean of normal pregnant women, instead of opting for the concentration derived from receiver operating curve to limit the false-positive rate in normal pregnant women. Although plasma sVEGFR-1 concentration is higher in pre-eclampsia than in the control group, approximately 2 months prior to the clinical manifestations the diagnostic indices are very poor. The optimal time to determine plasma sVEGFR-1 concentrations from a diagnostic/prognostic point of view is 28-32 weeks of gestation (mean 30 weeks) for early-onset pre- eclampsia, and 30-34 weeks of gestation (mean 32 weeks) for the late- onset disease, or approximately one month before the clinical diagnosis.

In conclusion, our study demonstrates that plasma sVEGFR-1 concentration is elevated in pre-eclampsia approximately 8 weeks before, and the increase is more pronounced at 2-5 weeks prior to the clinical manifestations of the disease. This observation may have clinical and therapeutic implications. Preventive strategies such as neutralization against sVEGFR-1 [139] or administration of pro-angiogenic factors (i.e., PlGF and VEGF) could be considered. However, it is noteworthy to emphasize that although these measures could prevent further endothelial cell damage and probably delay clinical hypertension and proteinuria, they may not correct utero- placental insufficiency. Thus, fetal death could still occur. Furthermore, it is crucial to maintain the balance between plasma sVEGFR-1 and other pro-angiogenic factors, since excess VEGF could, in turn, induce exaggerated vasodilatation and increased vessel permeability.

References

1. Romero R, Lockwood C, Oyarzun E, et al. Toxemia: new concepts in an old disease. Semin Perinatol 1988;12:302-323.

2. Redman CW, Sargent IL. The pathogenesis of pre-eclampsia. Gynecol Obstet Fertil 2001;29:518-522.

3. Dekker GA, Sibai BM. Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 1998;179:1359- 1375.

4. Roberts JM, Lain KY. Recent Insights into the pathogenesis of pre-eclampsia. Placenta 2002;23:359-372.

5. Sibai BM. Diagnosis and management of gestational hypertension and preeclampsia. Obstet Gynecol 2003;102:181-192.

6. Kaufmann P, Black S, Huppertz B. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod 2003;69:l-7.

7. Myatt L. Role of placenta in preeclampsia. Endocrine 2002;19:103-111.

8. Brosens JJ, Pijnenborg R, Brosens IA. The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature. Am J Obstet Gynecol 2002;187:1416-1423.

9. Norwitz ER, Hsu CD, Repke JT. Acute complications of preeclampsia. CHn Obstet Gynecol 2002;45:308-329.

10. Martin JN, Jr., Rinehart BK, May WL, et al. The spectrum of severe preeclampsia: comparative analysis by HELLP (hemolysis, elevated liver enzyme levels, and low platelet count) syndrome classification. Am J Obstet Gynecol 1999;180:1373-1384.

11. Robertson WB, Brosens I, Dixon G. Maternal uterine vascular lesions in the hypertensive complications of pregnancy. Perspect Nephrol Hypertens 1976;5:115-127.

12. Pijnenborg R, Anthony J, Davey DA, et al. Placental bed spiral arteries in the hypertensive disorders of pregnancy. Br J Obstet Gynaecol 1991;98:648-655.

13. Zhou Y, Damsky CH, Fisher SJ. Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotypc. One cause of defective endovascular invasion in this syndrome? J CHn Invest 1997;99:2152-2164.

14. Zhou Y, Fisher SJ, Janatpour M, et al. Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion? J CHn Invest 1997;99:2139-2151.

15. Roberts JM. Endothelial dysfunction in preeclampsia. Semin Reprod Endocrinol 1998;16:5-15.

16. Hunter CA Jr, Howard WF. A presser substance (hysterotonin) occurring in toxemia. Am J Obstet Gynecol 1960;79:838-846.

17. Zuspan FP. Catecholamines. Their role in pregnancy and the development of pregnancy-induced hypertension. J Reprod Med 1979;23:143-150.

18. Neale D, Demasio K, Illuzi J, et al. Maternal serum of women with pre-eclampsia reduces trophoblast cell viability: evidence for an increased sensitivity to Fasmediated apoptosis. J Matern Fetal Neonatal Med 2003;13:39-44.

19. Pirani BB, MacGillivray I. The effect of plasma retransfusion on the blood pressure in the puerperium. Am J Obstet Gynecol 1975;121:221-226.

20. Krege JH, Katz VL. A proposed relationship between vasopressinase altered vasopressin and preeclampsia. Med Hypotheses 1990;31:283-287.

21. McKinncy ET, Shouri R, Hunt RS, et al. Plasma, urinary, and salivary 8-epi-prostaglandin f2alpha levels in normotensive and preeclamptic pregnancies. Am J Obstet Gynecol 2000; 183:874-877.

22. Pedersen EB, Aalkjaer C, Christensen NJ, et al. Renin, angiotensin II, aldosterone, catecholamines, prostaglandins and vasopressin. The importance of presser and depressor factors for hypertension in pregnancy. Scand J Clin Lab Invest Suppl 1984; 169:48-56.

23. Conrad KP, Benyo DF. Placental cytokines and the pathogenesis of preeclampsia. Am J Reprod Immunol 1997;37:240-249.

24. Granger JP, Alexander BT, Llinas MT, et al. Pathophysiology of preeclampsia: linking placental ischemia/hypoxia with microvascular dysfunction. Microcirculation 2002;9:147160.

25. Zhou Y, McMaster M, Woo K, et al. Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am J Pathol 2002;160:1405-1423.

26. Zhou Y, Bellingard V, Feng KT, et al. Human cytotrophoblasts promote endothelial survival and vascular remodeling through secretion of Ang2, PlGF, and VEGF-C. Dev Biol 2003;263:114-125.

27. Ahmed A, Perkins J. Angiogenesis and intrauterine growth restriction. Baillieres Best Pract Res CHn Obstet Gynaecol 2000;14:981-998.

28. Charnock-Jones DS, Burton GJ. Placental vascular morphogenesis. Baillieres Best Pract Res Clin Obstet Gynaecol 2000;14:953-968.

29. Ong S, Lash G, Baker PN. Angiogenesis and placental growth in normal and compromised pregnancies. Baillieres Best Pract Res Clin Obstet Gynaecol 2000; 14:969-980.

30. Torry DS, Mukherjea D, Arroyo J, et al. Expression and function of placenta growth factor: implications for abnormal placentation. J Soc Gynecol Invest 2003; 10:178-188.

31. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669-676.

32. Gerber HP, McMurtrey A, Kowalski J, et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for FIk-I/KDR activation. J Biol Chem 1998;273:30336-30343.

33. Carmeliet P, Moons L, Luttun A, et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med 2001;7:575-583.

34. Mac Gabhann F, Popel AS. Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells. Am J Physiol Heart Circ Physiol 2003;286:H153-H164.

35. Hiratsuka S, Maru Y, Okada A, et al. Involvement of Fit-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. Cancer Res 2001 ; 61:1207-1213.

36. Athanassiades A, LaIa PK. Role of placenta growth factor (PIGF) in human extravillous trophoblast proliferation, migration and invasiveness. Placenta 1998;19:465-473.

37. Cheung CY. Vascular endothelial growth factor: possible role in fetal development and placental function. J Soc Gynecol Invest 1997;4:169-177.

38. Clark DE, Smith SK, He Y, et al. A vascular endothelial growth factor antagonist is produced by the human placenta and released into the maternal circulation. Biol Reprod 1998;59:1540- 1548.

39. Desai J, Holt-Shore V, Torry RJ, et al. Signal transduction and biological function of placenta growth factor in primary human trophoblast. Biol Reprod 1999;60:887-892.

40. Hayman R, Brockelsby J, Kenny L, et al. Preeclampsia: the endothelium, circulating factor(s) and vascular endothelial growth factor. J Soc Gynecol Invest 1999;6:3-10.

41. Hornig C, Barleon B, Ahmad S, et al. Release and complex formation of soluble VEGFR-I from endothelial cells and biological fluids. Lab Invest 2000;80:443-454.

42. Kendall RL, Wang G, Thomas KA. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT- I, and its heterodimerization with KDR. Biochem Biophys Res Commun 1996;226:324-328.

43. Kupferminc MJ, Daniel Y, Englender T, et al. Vascular endothelial growth factor is increased in patients with preeclampsia. Am J Reprod Immunol 1997;38:302-306.

44. Lash GE, Cartwright JE, Whitley GS, et al. The effects of angiogenic growth factors on extravillous trophoblast invasion and motility. Placenta 1999;20:661-667.

45. Lyall F, Greer IA. The vascular endothelium in normal pregnancy and pre-eclampsia. Rev Reprod 1996; 1:107-116.

46. Lyall F, Greer IA, Boswell F, et al. Suppression of serum vascular endothelial growth factor immunoreactivity in normal pregnancy and in pre-eclampsia. Br J Obstet Gynaecol 1997;104:223- 228.

47. Reuvekamp A, Velsing-Aarts FV, Poulina IE, et al. Selective deficit of angiogenic growth factors characterises pregnancies complicated by pre-eclampsia. Br J Obstet Gynaecol 1999; 106:1019- 1022.

48. Reynolds LP, Redmer DA. Utero-placental vascular development and placental function. J Anim Sci 1995;73:1839-1851.

49. Schlembach D, Beinder E. Angiogenic factors in preeclampsia. J Soc Gynecol Invest 2003;10:316A.

50. Taylor RN, de Groot CJ, Cho YK, et al. Circulating factors as markers and mediators of endothelial cell dysfunction in preeclampsia. Semin Reprod Endocrinol 1998; 16:17-31.

51. Taylor RN, Grimwood J, Taylor RS, e\t al. Longitudinal serum concentrations of placental growth factor: evidence for abnormal placental angiogenesis in pathologic pregnancies. Am J Obstet Gynecol 2003;188:177-182.

52. Tidwell SC, Ho HN, Chiu WH, et al. Low maternal serum levels of placenta growth factor as an antecedent of clinical preeclampsia. Am J Obstet Gynecol 2001;184:1267-1272.

53. Torry DS, Wang HS, Wang TH, et al. Preeclampsia is associated with reduced serum levels of placenta growth factor. Am J Obstet Gynecol 1998; 179:1539-1544.

54. Torry DS, Ahn H, Barnes EL, et al. Placenta growth factor: potential role in pregnancy. Am J Reprod Immunol 1999;41:79-85.

55. Tsatsaris V, Goffin F, Munaut C, et al. Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences. J Clin Endocrinol Metab 2003;88:5555-5563.

56. Vuorela P, Hatva E, Lymboussaki A, et al. Expression of vascular endothelial growth factor and placenta growth factor in human placenta. Biol Reprod 1997;56:489-494.

57. Maynard SE, Min JY, Merchan J, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFltl) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649-658.

58. Koga K, Osuga Y, Yoshino O, et al. Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-I) levels in women with preeclampsia. J Clin Endocrinol Metab 2003;88:2348-2351.

59. Chaiworapongsa T, Romera R, Espinoza J, et al. Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia. Young Investigator Award. Am J Obstet Gynecol 2004;190:1541-47.

60. Livingston JC, Chin R, Haddad B, et al. Reductions of vascular endothelial growth factor and placental growth factor concentrations in severe preeclampsia. Am J Obstet Gynecol 2000; 183:1554-1557.

61. Kincaid-Smith P. The renal lesion of preeclampsia revisited. Am J Kidney Dis 1991;17:144-148.

62. Hill PA, Fairley KF, Kincaid-Smith P, et al. Morphologic changes in the renal glomerulus and the juxtaglomerular apparatus inhuman preeclampsia. J Pathol 1988; 156:291-303.

63. Gaber LW, Spargo BH, Lindheimer MD. Renal pathology in pre- eclampsia. Baillieres Clin Obstet Gynaecol 1987;1:971-995.

64. Strevens H, Wide-Swensson D, Hansen A, et al. Glomerular endotheliosis in normal pregnancy and pre-eclampsia. BJOG 2003;110:831-836.

65. Sibai BM, Ewell M, Levine RJ, et al. Risk factors associated with preeclampsia in healthy nulliparous women. The Calcium for Preeclampsia Prevention (CPEP) Study Group. AmJ Obstet Gynecol 1997;177:1003-1010.

66. Levine RJ, Maynard SE, Qian C, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672- 683.

67. Gant NF, Chand S, Whalley PJ, et al. The nature of presser responsiveness to angiotensin II in human pregnancy. Obstet Gynecol 1974;43:854.

68. Gant NF, Chand S, Worley RJ, et al. A clinical test useful for predicting the development of acute hypertension in pregnancy. Am J Obstet Gynecol 1974; 120:1-7.

69. Harrington KF, Campbell S, Bewley S, et al. Doppler velocimetry studies of the uterine artery in the early prediction of pre-eclampsia and intra-uterine growth retardation. Eur J Obstet Gynecol Reprod Biol 1991;42 Suppl:S14-S20.

70. Kurmanavicius J, Florio I, Wisser J, et al. Reference resistance indices of the umbilical, fetal middle cerebral and uterine arteries at 24-42 weeks of gestation. Ultrasound Obstet Gynecol 1997;10:112-120.

71. Bower S, Schuchter K, Campbell S. Doppler ultrasound screening as part of routine antenatal scanning: prediction of pre- eclampsia and intrauterine growth retardation. Br J Obstet Gynaecol 1993;100:989-994.

72. Adamson SL, Morrow RJ, Bascom PA, et al. Effect of placental resistance, arterial diameter, and blood pressure on the uterine arterial velocity waveform: a computer modeling approach. Ultrasound Med Biol 1989; 15:437-442.

73. Ochi H, Suginami H, Matsubara K, et al. Micro-bead embolization of uterine spiral arteries and changes in uterine arterial flow velocity waveforms in the pregnant ewe. Ultrasound Obstct Gynecol 1995;6:272-276.

74. Ochi H, Matsubara K, Kusanagi Y, et al. Significance of a diastolic notch in the uterine artery flow velocity waveform induced by uterine embolisation in the pregnant ewe. Br J Obstet Gynaecol 1998;105:1118-1121.

75. Talbert DG. Uterine flow velocity waveform shape as an indicator of maternal and placenta! development failure mechanisms: a model-based synthesizing approach. Ultrasound Obstet Gynecol 1995;6:261-271.

76. Lin S, Shimizu I, Suehara N, et al. Uterine artery Doppler velocimetry in relation to trophoblast migration into the myometrium of the placental bed. Obstet Gynecol 1995;85:760-765.

77. Olofsson P, Laurini RN, Marsal K. A high uterine artery pulsatility index reflects a defective development of placental bed spiral arteries in pregnancies complicated by hypertension and fetal growth retardation. Eur J Obstet Gynecol Reprod Biol 1993;49:161- 168.

78. Campbell S, Black RS, Lees CC, et al. Doppler ultrasound of the maternal uterine arteries: disappearance of abnormal waveforms and relation to birthweight and pregnancy outcome. Acta Obstet Gynecol Scand 2000;79:631-634.

79. Albaiges G, Missfelder-Lobos H, Lees C, et al. One-stage screening for pregnancy complications by color Doppler assessment of the uterine arteries at 23 weeks' gestation. Obstet Gynecol 2000;96:559-564.

80. Chappell L, Bewley S. Pre-eclamptic toxaemia: the role of uterine artery Doppler. BrJ Obstet Gynaecol 1998;105:379-382.

81. Zimmermann P, Eirio V, Koskinen J, et al. Doppler assessment of the uterine and uteroplacental circulation in the second trimester in pregnancies at high risk for preeclampsia and/or intrauterine growth retardation: comparison and correlation between different Doppler parameters. Ultrasound Obstet Gynecol 1997;9:330- 338.

82. Todros T, Ferrazzi E, Arduini D, et al. Performance of Doppler ultrasonography as a screening test in low risk pregnancies: results of a multicentric study. J Ultrasound Med 1995; 14:343-348.

83. North RA, Ferrier C, Long D, et al. Uterine artery Doppler flow velocity waveforms in the second trimester for the prediction of preeclampsia and fetal growth retardation. Obstet Gynecol 1994;83:378-386.

84. Schulman H. The clinical implications of Doppler ultrasound analysis of the uterine and umbilical arteries. Am J Obstet Gynecol 1987; 156:889-893.

85. Fleischer A, Schulman H, Farmakides G, et al. Uterine artery Doppler velocimetry in pregnant women with hypertension. Am J Obstet Gynecol 1986;154:806-813.

86. Papageorghiou AT, Yu CK, Bindra R, et al. Multicenter screening for pre-eclampsia and fetal growth restriction by transvaginal uterine artery Doppler at 23 weeks of gestation. Ultrasound Obstet Gynecol 2001;18:441-449.

87. Ahmed A, Li XF, Dunk C, et al. Colocalisation of vascular cndothelial growth factor and its Fit-1 receptor in human placenta. Growth Factors 1995;12:235-243.

88. Charnock-Jones DS, Sharkey AM, Boocock CA, et al. Vascular cndothelial growth factor receptor localization and activation in human trophoblast and choriocarcinoma cells. Biol Reprod 1994;51:524- 530.

89. Clark DE, Smith SK, Sharkey AM, et al. Localization of VEGF and expression of its receptors fit and KDR in human placenta throughout pregnancy. Hum Reprod 1996;! 1:1090-1098.

90. Khaliq A, Dunk C, Jiang J, et al. Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up- regulates placenta growth factor expression: molecular evidence for "placental hyperoxia" in intrauterine growth restriction. Lab Invest 1999;79:151-170.

91. Lash GE, Taylor CM, Trew AJ, et al. Vascular endothelial growth factor and placental growth factor release in cultured trophoblast cells under different oxygen tensions. Growth Factors 2002;20:189-196.

92. Shore VH, Wang TH, Wang CL, et al. Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast. Placenta 1997; 18:657-665.

93. Helske S, Vuorela P, Carpen O, et al. Expression of vascular endothelial growth factor receptors 1, 2 and 3 in placentas from normal and complicated pregnancies. MoI Hum Reprod 2001;7:205-210.

94. Kumazaki K, Nakayama M, Suehara N, et al. Expression of vascular endothelial growth factor, placental growth factor, and their receptors Fit-1 and KDR in human placenta under pathologic conditions. Hum Pathol 2002;33:1069-1077.

95. Trollmann R, Amann K, Schoof E, et al. Hypoxia activates the human placental vascular endothelial growth factor system in vitro and in vivo: up-regulation of vascular endothelial growth factor in clinically relevant hypoxic ischemia in birth asphyxia. Am J Obstet Gynecol 2003;188:517-523.

96. Ahmed A, Dunk C, Kniss D, et al. Role of VEGF receptor-1 (Fit- 1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells. Lab Invest 1997;76:779- 791.

97. Adianassiades A, Hamilton GS, LaIa PK. Vascular endothelial growth factor stimulates proliferation but not migration or invasiveness in human extravillous trophoblast. Biol Reprod 1998;59:643-654.

98. Benirschke K, Kaufmann P. Pathology of the human placenta. 4th ed. New York: Springer; 2000. pp 116-154.

99. Kingdom JC, Kaufmann P. Oxygen and placental vascular development. Adv Exp Med Biol 1999;474:259-275.

100. Todros T, Sciarrone A, Piccoli E, et al. Umbilical Doppler waveforms and placental villous angiogenesis in pregnancies complicated by fetal growth restriction. Obstet Gynecol 1999;93:499- 503.

101. Kingdom J, Huppertz B, Seaward G, et al. Development of the placental villous tree and its consequences for fetal growth. Eur J Obstet Gynecol Reprod Biol 2000;92:35-43.

102. Geva E, Ginzinger DG, Zaloudek CJ, et al. Human placental vascular development: vasculogenic and angiogenic (branching and nonbranching) transformation is regulatedby vascular endothelial growth factor-A, angiopoietin-1, and angiopoietin-2. J Clin Endocrinol Metab 2002;87:4213-4224.

103. Mayhew TM. Fetoplacental angiogenesis during gestation is biphasic, longitudinal and occurs by proliferation and remodelling of vascular endothelial cells. Placenta 2002;23:742-750.

104. Kaufmann P, Luckhardt M, Schweikhart G, et al. Crosssectional features and three-dimensional structure of human placental villi. Placenta 1987;8:235-247.

105. Jackson MR, Walsh AJ, Morrow RJ, et al. Reduced placental villous tree elaboration in small-for-gestational-age pregnancies: relationship with umbilical artery Doppler waveforms. Am J Obstet Gynecol 1995;172:518-525.

106. Krebs C, Macara LM, Leiser R, et al. Intrauterine growth restriction with absent end-diastolic flow velocity in the umbilical artery is associated with maldevelopment of the placental terminal villous tree. Am J Obstet Gynecol 1996;175:1534-1542.

107. Boyle JW, Lotgering FK, Longo LD. Acute embolization of the uteroplacental circulation: uterine blood flow and placental CO diffusing capacity. J Dev Physiol 1984;6:377386.

108. Brar HS, Medearis AL, DeVore GR, et al. A comparative study of fetal umbilical velocimetry with continuous- and pulsed-wave Doppler ultrasonography in high-risk pregnancies: relationship to outcome. Am J Obstet Gynecol 1989;160:375-378.

109. Trudinger BJ, Cook CM. Doppler umbilical and uterine flow waveforms in severe pregnancy hypertension. Br J Obstet Gynaecol 1990;97:142-148.

110. Trudinger BJ, Cook CM, Giles WB, et al. Fetal umbilical artery velocity waveforms and subsequent neonatal outcome. Br J Obstet Gynaecol 1991;98:378-384.

111. Kuzuya M, Ramos MA, Kanda S, et al. VEGF protects against oxidized LDL toxicity to endothelial cells by an intracellular glutathione-dependent mechanism through the KDR receptor. Arterioscler Thromb Vase Biol 2001;21:765-770.

112. Horowitz JR, Rivard A, van der ZR, et al. Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium. Arterioscler Thromb Vase Biol 1997; 17:2793-2800.

113. Polliotti BM, Fry AG, Sailer DN, et al. second-trimester maternal serum placental growth factor and vascular endothelial growth factor for predicting severe, early-onset preeclampsia. Obstet Gynecol 2003;101:1266-1274.

114. Su YN, Lee CN, Cheng WF, et al. Decreased maternal serum placenta growth factor in early second trimester and preeclampsia. Obstet Gynecol 2001;97:898-904.

115. Tjoa ML, van Vugt JM, Mulders MA, et al. Plasma placenta growth factor levels in midtrimester pregnancies. Obstet Gynecol 2001;98:600-607.

116. Yang S, Toy K, Ingle G, et al. Vascular endothelial growth factor-induced genes in human umbilical vein endothelial cells: relative roles of KDR and Fit-1 receptors. Arterioscler Thromb Vase Biol 2002;22:1797-1803.

117. Kroll J, Waltenberger J. VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR). Biochem Biophys Res Commun 1998;252:743-746.

118. Kroll J, Waltenberger J. A novel function of VEGF receptor- 2 (KDR): rapid release of nitric oxide in response to VEGF-A stimulation in endothelial cells. Biochem Biophys Res Commun 1999;265:636-639.

119. Tsurumi Y, Murohara T, Krasinski K, et al. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nat Med 1997;3:879-886.

120. He H, Venema VJ, Gu X, et al. Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src. J Biol Chem 1999;274:25130-25135.

121. Gliki G, Abu-Ghazaleh R, Jezequel S, et al. Vascular endothelial growth factor-induced prostacyclin production is mediated by a protein kinase C (PKC)-dependent activation of extracellular signal-regulated protein kinases 1 and 2 involving PKC- delta and by mobilization of intracellular Ca[2]^sup +^ . Biochem J 2001;353:503-512.

122. Sugimoto H, Hamano Y, Charytan D, et al. Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J Biol Chem 2003;278:12605-12608.

123. Ostendorf T, Kunter U, Eitner F, et al. VEGF(165) mediates glomerular endothelial repair. J Clin Invest 1999;104:913-923.

124. Kabbinavar F, Hurwitz HI, Fehrenbacher L, et al. Phase II, randomized trial comparing bevacizumab plus fluorouracil (FUyieucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J CHn Oncol 2003;21:60-65.

125. Bayhan G, Atamer Y, Atamer A, et al. Significance of changes in lipid peroxides and antioxidant enzyme activities in pregnant women with preeclampsia and eclampsia. Clin Exp Obstet Gynecol 2000;27:142-146.

126. Mutlu-Turkoglu U, Aykac-Toker G, Ibrahimoglu L, et al. Plasma nitric oxide metabolites and lipid peroxide levels in preeclamptic pregnant women before and after delivery. Gynecol Obstet Invest 1999;48:247-250.

127. Winkler K, Wetzka B, Hoffmann MM, et al. Triglyceride-rich lipoproteins are associated with hypertension in preeclampsia. J Clin Endocrinol Metab 2003;88:1162-1166.

128. Takacs P, Kauma SW, Sholley MM, et al. Increased circulating lipid peroxides in severe preeclampsia activate NF-kappaB and upregulate ICAM-I in vascular endothelial cells. FASEB J 2001;15:279- 281.

129. Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med 1999;222:222-235.

130. Vaughan JE, Walsh SW. Oxidative stress reproduces placental abnormalities of preeclampsia. Hypertens Pregnancy 2002;21:205-223.

131. Myatt L, Kossenjans W, Sa

Source: Journal of Maternal - Fetal & Neonatal Medicine

More News in this Category