Conceived and designed the experiments: AAK SM EJ. Performed the experiments: AAK. Analyzed the data: AAK. Contributed reagents/materials/analysis tools: SM EJ. Wrote the paper: AAK SM KH EJ.
The authors have declared that no competing interests exist.
Antihypertensive drugs are believed to lower blood pressure in pre-eclampsia by direct or central vasodilatory mechanisms. However, they could also act by decreasing production of anti-angiogenic proteins involved in the pathophysiology of hypertension and proteinuria in pre-eclampsia (PE). The aim of our study was to evaluate the impact of antihypertensive therapy with alpha methyldopa on maternal circulating levels and placental production of soluble fms-like tyrosine kinase 1 (sFlt-1), soluble endoglin (sEng), vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in hypertensive disorders of pregnancy.
In a study conducted at University College Hospital and the Homerton University Hospital in London, we recruited 51 women with PE, 29 with gestational hypertension (GH), and 80 matched normotensive controls. Eight (16%) of the women with PE had severe disease. Placental samples were obtained from a further 48 women (14 PE, 10 GH and 24 matched controls). Serum levels of angiogenic factors were measured before and 24–48 hours after commencing antihypertensive therapy with alpha methyldopa for clinical indications. The same parameters were measured in placental extracts. In both PE (
Our data suggest that alpha methyldopa may have a specific effect on placental and/or endothelial cell function in pre-eclampsia patients, altering angiogenic proteins.
Anti-angiogenic and pro-angiogenic factors are known to play an important role in the pathophysiology of pre-eclampsia (PE)
Soluble Flt-1 is a splice variant of VEGF receptor 1 (Flt-1) which is produced by a variety of tissues. Investigation of uterine vein levels of sFlt-1 at cesarean section in pre-eclampsia has suggested a uterine source
The most commonly used drug for the treatment of hypertensive disorders in pregnancy in the UK is alpha methyldopa (αMD). Alpha methyldopa acts on alpha-2 adrenoreceptors and is believed to exert its antihypertensive effect primarily in the central nervous system
Belgore et al
Serum and placental tissue samples were obtained over an 18 month period from women with singleton pregnancies prospectively recruited in the second and third trimesters of pregnancy at the Homerton University Hospital, London. During this period, approximately 6,000 deliveries took place. Demographic and clinical data including age, body mass index (BMI), parity, blood pressure (BP) and gestational age (GA) were recorded. Gestational age was established on the basis of menstrual date and/or ultrasonographic examination prior to 20 weeks of gestation.
All women were followed up until after delivery, and fetal and maternal outcomes were obtained from the women's medical records and labour ward records. Written consent was obtained from each woman after receiving full written information about the research project. This study was approved by the Camden & Islington Community Local Research Ethics Committee and The University College London Hospitals Committee on the Ethics of Human Research. Exclusion criteria included multiple pregnancy, history of hypertension, diabetes, renal disease or immune disorders or women taking medication which could affect blood pressure.
The study group in whom serum levels were measured included 51 women presenting with PE, 29 with gestational hypertension and 80 controls (
Flow diagrams of women recruited to the serum (a) and placental (b) arms of the study respectively.
Another group of 48 women were recruited for measurement of placental levels: 24 with hypertensive disorders in pregnancy (14 PE, 10 gestational hypertension), and 24 controls matched for maternal age, gestational age and parity (
BP was measured in duplicate using a standard mercury sphygmomanometer and the average of two readings taken. All readings were taken by the same investigator (AK) with the subject in the sitting position. Korotkoff sounds 1 and 5 were used to define systolic and diastolic BP respectively. Mean BP was calculated as diastolic BP+1/3 pulse pressure. PE was defined according to the guidelines of the International Society for the Study of Hypertension in Pregnancy
Severe pre-eclampsia was defined as severe hypertension (diastolic blood pressure ≥110 mmHg) and mild proteinuria, or mild hypertension and severe proteinuria (a 24-hour urine sample that contained ≥3.5 g protein or a urine specimen ≥3+ protein by dipstick measurement). Patients with an abnormal liver function test (aspartate aminotransferase >70 IU/L) and thrombocytopenia (platelet count <100,000/cm3) were also classified as having severe pre-eclampsia. Gestational hypertension (GH) was defined as a diastolic blood pressure ≥90 mm Hg on at least two consecutive occasions in the second half of pregnancy, without proteinuria, in a previously normotensive woman
We collected blood samples at similar gestational periods (±4 days) from the hypertensive and matched control participants. All women in the control group had uncomplicated pregnancies. They had no history of cardiovascular disease, hypertension, proteinuria or fetal growth restriction, and were not taking medication that could affect blood pressure. The diastolic blood pressure of all women with PE or gestational hypertension was higher than 95 mm Hg. They received oral antihypertensive therapy in the form of alpha methyldopa 750–1500 mg/day for clinical indications according to local clinical protocols. In accordance with local protocols, co-existing fetal growth restriction did not influence the decision to institute antihypertensive therapy.
Venous blood was collected before and after (24–48 hours) antihypertensive therapy was commenced. A single venous blood sample was collected from controls. The samples were centrifuged at 3,000 rpm for ten minutes. The serum was separated and frozen at −80°C for subsequent analysis.
Placental samples were collected from another group of women (n = 48) undergoing cesarean section (CS) before the onset of labour, including 14 presenting with PE, 10 with gestational hypertension (GH) and 24 normotensive controls matched for gestational age, maternal age (±3 years) and parity (none or one to two deliveries), and who were delivered by CS for obstetric reasons other than hypertension (e.g. preterm labour with an abnormal presentation or breech presentation at term). The hypertensive group included 12 women who received antenatal antihypertensive therapy (alpha methyldopa 750–1500 mg/day). They were matched for gestational age with 12 women who were not taking antihypertensive treatment. Four to five placental biopsies were obtained at random from the maternal surface of the placenta, free of placental membranes.
Using commercially available kits for the measurement of human sFlt-1, PlGF, free VEGF and sEng (R & D Systems, Minneapolis, Minnesota, USA), two-site enzyme linked immunosorbent assays (ELISAs) were conducted in duplicate according to the manufacturer's protocol. The minimum detectable levels for serum sFlt-1, PlGF, VEGF and sEng were 5 pg/ml, 7 pg/ml, 9 pg/ml and 7 pg/ml respectively. The VEGF level in the serum samples was below the detectable levels. Intra- and inter-assay co-efficients of variation respectively in our laboratory were as follows: serum sFlt-1 7%, 9%; PlGF 5%, 6%; and sEng 6%, 8%.
The samples (placental villi, 164–559 mg wet weight) were rinsed in sterile phosphate buffered saline and snap frozen. A known weight of placental biopsy was homogenized manually in four volumes (w/v) of Tris buffered saline containing EDTA-free serine-/cysteine-protease inhibitor cocktail, diluted according to the manufacturer's instructions (Roche Biochemicals, Indiana, USA) using a homogenizer. The protein extract was collected after centrifugation (3,000 rpm for 10 minutes) and stored at −80°C until quantitative analyses were performed in batches.
The total protein concentration was determined with the Bradford assay (Pierce, Lausanne, Switzerland) using human serum albumin as a standard. Commercial ELISAs from R&D systems (Minneapolis, Minnesota, USA) were used to measure sFlt-1, PlGF, sEng and ‘free’ VEGF in placental extracts according to the manufacturer's protocol. All samples were assayed in duplicate and the intra- and inter-assay variations were <12% for all assays. The minimum detectable limits for the assays were: sFlt-1: 31.3pg/ml; PlGF: 3.9 pg/ml; sEng: 62.5 pg/ml; and free VEGF 7.8 pg/ml. These assays were validated for placental samples. Placental samples from cases with PE were diluted parallel to the standard curve in the sFlt-1, PlGF, sEng and VEGF ELISAs.
Uterine artery Doppler pulsatility index (PI) was measured in the hypertensive and control groups at the time of recruitment. In women who received antihypertensive therapy, Doppler measurements were taken before and after (24–48 hours) therapy was initiated. The uterine artery was identified by a combination of real-time and colour Doppler techniques (iU22 Ultrasound System, Philips Medical Systems, Bothell, WA, USA). Blood velocity waveforms were recorded by the pulsed Doppler method (3.5 MHz curved probe; 120 Hz high-pass filter). Five consecutive flow velocity waveforms of good quality were recorded, and the mean pulsatility index (PI) derived.
D'Agostino and Pearson Omnibus test was used to assess normality of continuous data. Analysis of variance (ANOVA) with Bonferroni post hoc tests were carried out to study the differences among the three groups. Data were analyzed in two gestational age intervals: <34 weeks representing early-onset disease and ≥34 weeks representing late-onset disease. Paired
<34+0 weeks | ≥34+0 weeks | |||||||
Controls | PE | GH | Controls | PE | GH | |||
n | 41 | 28 | 13 | 39 | 23 | 16 | ||
Age (years) |
31±4 | 30±5 | 32±4 | 0.6 | 31±4 | 30±4 | 32±5 | 0.2 |
BMI (kg/m2) |
27±4 | 30±4 | 27±4 | 0.07 | 28±3 | 27±5 | 29±5 | 0.2 |
Nulliparity [n (%)] | 25 (61) | 20 (71) | 7 (54) | 0.6 | 21 (53) | 16 (70) | 9 (56) | 0.3 |
Current smoker [n (%)] | 1 (2) | 0 (0) | 0 (0) | 1 | 2 (5) | 1 (4) | 0 (0) | 1 |
Caucasian [n (%)] | 20 (48) | 13 (46) | 7 (54) | 0.7 | 23 (59) | 13 (57) | 9 (56) | 0.4 |
GA at recruitment (days) |
30±1.3 | 30±0.4 | 30.4±0.8 | 0.5 | 36.6±2.4 | 36±2.3 | 36.4±2 | 0.4 |
GA at delivery (days) |
39.7±2.3 | 33±1.7 | 36.7±2.9 | <0.001 | 39.8±1.6 | 37.3±2 | 38.6±2.3 | <0.001 |
Birth weight (grammes) |
3398±529 | 1685±204 | 2725±198 | <0.001 | 3405±526 | 2896±689 | 3174±591 | <0.001 |
Mean BP (mmHg) | 85±12 | 126.6±12 | 125.1±12 | <0.0001 | 85±11 | 121.1±11.1 | 114.5±6.1 | <0.0001 |
Mean Pulsatility Index (PI) | 0.76±0.1 | 1.51±0.5 | 0.84±0.1 | <0.0001 | 0.59±0.1 | 0.85±0.2 | 0.61±0.1 | <0.0001 |
Interval between samples (hours) | 32±4 | 34±3 | 38±6 | 39±7 |
BMI = body mass index.
GA = gestational age.
Mean BP = mean blood pressure.
PE = pre-eclampsia.
GH = gestational hypertension.
Data presented as mean±SD and analyzed by one-way ANOVA with Bonferroni post hoc analysis.
Mean serum sFlt-1 (a), PlGF (b) and sEng (c) concentrations in normotensives (controls), women with pre-eclampsia and women with gestational hypertension according to gestational age (GA) interval [early onset <34 weeks (41 controls, 28 PE, 13 GH) and late onset ≥34 weeks (39 controls, 23 PE, 16 GH)]. Error bars represent standard errors. Comparison of controls and cases (with PE or GH) was performed after logarithmic transformation. Levels before and after alpha methyldopa therapy are shown for women with pre-eclampsia and women with gestational hypertension. The levels in the three groups were compared using ANOVA with Bonferroni Dunn's posthoc tests. The levels before and after antihypertensive therapy are compared using the paired
Mean maternal serum concentrations of sFlt-1 (a), PlGF (b) and soluble endoglin (c) in women with early onset and late onset PE, and in women with mild and severe PE. Error bars represent standard errors. Early onset were compared with late onset, and mild with severe PE after logarithmic transformation using unpaired
The results of the placental analysis are presented in
Concentrations of sFlt-1(a), PlGF (b), soluble endoglin (c) and VEGF (d) (expressed per mg protein) in placental tissue from normotensive (controls, n = 24, pre-eclampsia (PE, n = 14) and gestational hypertension (HT, n = 10 pregnancies. Error bars represent standard errors. Comparison of controls and cases (with PE or GH) was performed after logarithmic transformation. The mean gestational ages (days) for the three groups were (mean±SD): controls 242±16; pre-eclampsia 238±13; gestational hypertension 243±20.
PE | GH | Controls | |||||
Anti-hypertensive (n = 7) | No anti-hypertensive (n = 7) | Anti-hypertensive (n = 5) | No anti-hypertensive (n = 5) | ||||
sFlt-1 (ng/ml) | 17.7 (3) |
33.9 (5) |
0.01 | 3.1 (0.4) | 5.3 (0.8) | 0.06 | 5.2 (1.2) |
PlGF (pg/ml) | 8.4 (1.8) |
7.7 (2) |
0.56 | 20.7 (3) | 15.9 (4) | 0.18 | 23.9 (5) |
sEng (ng/ml) | 13.1 (1.6) |
19.6 (1.7) |
0.02 | 3.8 (0.4) | 4.3 (0.3) | 0.35 | 3.5 (0.2) |
VEGF (pg/ml) | 107.5 (30) |
247 (82) |
0.09 | 108 (39) |
166 (32) |
0.1 | 10.4 (1.2) |
Placental concentrations of sFlt-1, PlGF, sEng and VEGF (expressed per mg protein) in normotensive controls, pre-eclampsia (PE) and gestational hypertension (GH), grouped according to whether they received antihypertensive therapy or not. The mean gestational ages for the three groups were (mean±SD): controls 242±16; pre-eclampsia 238±13; gestational hypertension 243±20. The
PE = pre-eclampsia.
GH = gestational hypertension.
Data presented as mean (standard error of the mean), and analyzed by independent
There was no significant difference in mean uterine artery Doppler pulsatility index before and after methyldopa treatment, in either the PE (<34 weeks,
Pre-eclampsia remains one of the most complex disorders of human pregnancy. The lack of suitable animal models with placental features of the disease means that we have to rely, for the most part, on human studies. The maternal response to the presence of a pregnancy and placental activity remain the focus of research into the disease. The data from this study confirm that, in both early and late onset PE, maternal serum levels of sFlt-1 and sEng are higher, and PlGF lower, in women presenting with PE
Our data suggest that, in pre-eclampsia, placental concentrations of sFlt-1, sEng and PlGF mirror the maternal serum changes. These findings are consistent with the view that the placenta is the main source of sFlt-1, sEng and PlGF during pregnancy
Circulating sFlt-1 can bind to PlGF and VEGF, effectively inhibiting their actions
Our findings indicate that antihypertensive treatment with alpha methyldopa is associated with a significant fall in
Alpha methyldopa acts on α2-adrenergic receptors, primarily in the central nervous system (CNS) although an effect on peripheral α2-adrenoreceptors may also play a part
In 2007, Muthig et al demonstrated that down-regulation of α2β-adrenoceptors in mice placenta resulted in increased levels of Flt-1 and sFlt-1,
Due to the rapid evolution of hypertensive diseases in our study groups, we could investigate only the biological effects of the antihypertensive treatment over a short time interval (maximum 48 hours). Compared to long-term studies in non-pregnant women
A potential limitation of our study is the short time interval (24 to 48 hours) from initiation of antihypertensive treatment to venous blood sampling. It would be interesting to investigate the effect on angiogenic markers levels at longer intervals, e.g. a week after starting treatment. However, most women with hypertensive disorders in pregnancy, and particularly PE, will need delivery soon after starting antihypertensives, such that long-term follow-up is often precluded.
Our findings suggest that any future research into the use of serum markers to screen or monitor hypertensive disorders of pregnancy should take account of possible effects of antihypertensive therapy on marker levels. Further research is needed to evaluate whether different antihypertensive drugs have different effects on anti-angiogenic factors. Such research will improve our understanding of the pathophysiology of pre-eclampsia but may also lead to better therapeutic clinical protocols. Raised maternal serum levels of sFlt-1 can be detected several weeks prior to the onset of clinical pre-eclampsia. It is worth investigating whether administration of α-methyldopa at this point might have an effect on levels of anti-angiogenic factors and modify the disease process. Our findings also have potential implications outside the specialty of obstetrics. Women who develop pre-eclampsia are at significantly increased risk, later in life, of cardiovascular disease such as ischemic heart disease and stroke. Within this context, it is not known whether the use of specific antihypertensive drugs can also have a long-term beneficial effect. Furthermore, it remains to be determined whether the use of these antihypertensive drugs outside pregnancy could have a similar beneficial effect on anti-angiogenic factors and subsequently translate into clinical benefit. We hope that our data will stimulate further research in these areas.
It is not yet clear whether sFlt-1 and sEng are directly involved in the pathophysiology of PE or are simply markers of the disease process. Our data showing that antihypertensive treatment with alpha methyldopa is associated with a significant fall in their concentrations in both maternal serum and placenta is consistent with a positive effect on the control of disease progress. This finding supports the concept that pre-eclampsia combines an excessive maternal response to the presence of a pregnancy and placenta and progressive utero-placental insufficiency during the second half of pregnancy at the time of maximal fetal growth.