Objective
Patients with preeclampsia (PE) have endothelial dysfunction and an increased future risk of cardiovascular (CV) mortality. The number of circulating endothelial cells (CECs) is markedly increased in conditions associated with a high degree of endothelial cell activation/injury including PE. We hypothesized that the number of CECs continues to be increased in women with a history of PE, reflecting ongoing endothelial cell activation/injury.
Study Design
CECs, flow-mediated vasodilation, levels of adhesion molecules and soluble vascular endothelial growth factor receptor-1 (sVEGFR1), and urine albumin/creatinine ratio were determined in 21 healthy women with ongoing normal pregnancy, 24 healthy currently nonpregnant women with a history of normal pregnancy, a total of 17 women with currently active mild (n = 11) or severe (n = 6) PE without hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, and 16 currently nonpregnant women with a history of mild (n = 10) or severe (n = 6) PE.
Results
Blood samples from women with active preeclampsia had higher CECs (9.9 ± 7.9 cells/mL) than healthy pregnant women (3.0 ± 4.1 cells/mL; P < .001), healthy nonpregnant women with a history of normal pregnancy (3.4 ± 4.0 cells/mL; P < .001), or women with a history of preeclampsia (2.4 ± 2.0 cells/mL; P < .001). The number of CECs were similar between women with a history of preeclampsia and healthy nonpregnant women with a history of normal pregnancy. Patients with active preeclampsia had significantly higher soluble vascular cell adhesion molecule-1, soluble E-selectin, sVEGFR1, and urinary albumin/creatinine ratio than healthy pregnant women. However, soluble vascular cell adhesion molecule-1, soluble E-selectin, urinary albumin/creatinine ratio were similar in women with a history of preeclampsia and healthy nonpregnant women with a history of normal pregnancy. However, women with a history of preeclampsia had higher sVEGFR1 levels than women with a history of normal pregnancy ( P < .05).
Conclusion
Markers of endothelial activation, dysfunction, and damage were increased in patients with PE. After the delivery, this activation status is similar to the age-matched nonpregnant women with a history of normal pregnancy. However, sVEGFR-1 levels remain higher in women with a history of preeclampsia compared with women without a history of preeclampsia.
Preeclampsia is a major cause of maternal and neonatal mortality worldwide. It is associated with increased systemic vascular resistance, enhanced platelet aggregation, activation of the coagulation system, and endothelial cell dysfunction.
An initiating event in preeclampsia has been postulated to be reduced placental perfusion that leads to widespread dysfunction of the maternal vascular endothelium. Increased levels of soluble vascular endothelial growth factor receptor-1 (sVEGFR1) bind to the circulating vascular endothelial growth factor (VEGF) and prevents VEGF from binding to endogenous receptors, with a subsequent negative impact on angiogenesis.
Maynard et al have shown that sVEGFR1 levels are increased in the sera of preeclamptic subjects. When sVEGFR1 was removed from the preeclamptic placenta or VEGF was administered to block excess sVEGFR1, the antiangiogenic state returned to normal. The levels of cellular fibronectin, vascular cell adhesion molecule-1, intracellular cell adhesion molecule (ICAM)-1, E-selectin, CD31, and von Willebrand factor have been documented to be elevated in preeclamptic patients, even before its clinical manifestation.
Whereas structural changes return to normal after the termination of pregnancy, endothelial dysfunction and microalbuminuria may not resolve. Some studies have shown a higher incidence of microalbuminuria in women with a history of preeclampsia. Epidemiological studies provide evidence that women with a history of preeclampsia have a higher risk of cardiovascular complications and mortality.
In recent years, circulating endothelial cells (CECs) have emerged as markers of vascular damage. Although present in very small numbers in healthy individuals, their number increases dramatically in diseases with vascular damage, such as cardiovascular disease, specific infections, vasculitis, and type 2 diabetes. Furthermore, the number of CECs was an independent determinant of future cardiovascular mortality in patients with acute coronary events and in patients undergoing chronic hemodialysis treatment. In our study we aimed to determine whether endothelial activation/damage continues in women with a history of preeclampsia.
Materials and Methods
Study populations
The study was approved by the Ethics Committee of Marmara University Medical School and was carried out in accordance with the Declaration of Helsinki. All subjects gave informed consent for participation to the study.
The study population consisted of 4 groups: (1) the normal pregnancy group, consisting of 21 currently pregnant normotensive healthy women, with no proteinuria, referred to our obstetrics clinics; (2) the preeclampsia group, consisting of 17 pregnant subjects with currently active mild (n = 11) or severe (n = 6) PE and hospitalized; (3) the previous normal pregnancy group, consisting of 24 healthy nonpregnant women, who gave normal birth with no history of preeclampsia and proteinuria and are currently normotensive; and (4) the previous preeclampsia group, consisting of 16 nonpregnant women with a history of mild (n = 10) or severe (n = 6) PE in their previous pregnancies and whose last delivery was at least 6 months before enrollment into the study.
Preeclampsia was defined according to the following American College of Obstetricians and Gynecologists criteria: (1) measurement of a systolic blood pressure above 140 mm Hg and/or a diastolic blood pressure above 90 mm Hg in at least 2 measurements performed 6 hours apart after gestation week 20 in women who were previously normotensive, (2) new onset of proteinuria (300 mg/d) and greater protein elimination in the 24 hour urine or +1 or greater protein with a dipstick, and (3) resolution of proteinuria and hypertension within 12 weeks’ postpartum.
None of the controls from the normal pregnancy and previous normal pregnancy groups had hypertension, renal disease, or infection. Other exclusion criteria for the study populations were as follows: (1) any systemic disease that is known to cause endothelial dysfunction such as diabetes mellitus, chronic kidney disease, hyperlipidemia, atherosclerotic disesases, inflammatory or infectious processes within the last 3 months, and malignancy; (2) invasive procedures within the last month; (3) medications that can disrupt endothelial function such as aspirin or antilipidemic agents; (4) smoking; (5) multiple pregnancy; and (6) hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome.
Collection of blood and urine specimens
Blood specimens were collected from the study population in their midfollicular phase of the menstrual cycle except for the preeclamptic group. The blood samples from the preeclamptic group were collected immediately before the delivery. The first 8 mL of blood samples were withdrawn into serum-separating tubes for the separation and storage of serum and then another 5 mL into EDTA-containing tubes for the isolation of CECs. Early-morning urine samples were collected from the study population. All of the serum and urine samples were stored at –80°C in nonadsorbable tubes.
Isolation and enumeration of CECs
To isolate and enumerate CECs, we used the study protocol described previously. Briefly, 1 mL of venous EDTA-blood was blocked by an Fc receptor blocking agent (Miltenyi, Gladbach, Germany). AntiCD146-coated M-450 paramagnetic particles (Dynabeads; Dynal, Oslo, Norway) were then added and mixed in a head-over-head mixer for 30 minutes at 4°C and washed with phosphate-buffered saline/bovine serum albumin buffer in front of a magnet (Dynal, MPC-L; Dynal). Isolated cells were further stained with fluorescein isothiocyanate-coupled ulex europaeus agglutinin-1 solution (Sigma-Aldrich, St. Louis, MO) to confirm the endothelial origin of the cells. After washing, the CECs were counted under fluorescence microscopy using a Nageotte chamber. The CECs were identified according to the criteria defined by Woywodt et al.
Measurement of flow-mediated dilation
Flow – mediated dilation was measured as previously described. Measurements were taken in the fasting state before obtaining blood samples with the subjects resting for at least 10 minutes in the supine position. Measurements were done at end diastole by a single operator. We used a vascular ultrasound (Vivid 7 Dimension; GE Healthcare, Indianapolis, IN) with a 10 Mhz probe. The intraobserver variability was less than 5%. Percentage change in flow-mediated vasodilation (FMD) was calculated as the percentage changes in diameter relative to the baseline measurements.
Laboratory tests
Levels of serum soluble E-selectin, soluble intracellular cell adhesion molecule (sICAM)-1, soluble vascular cell adhesion molecule-1 (sVCAM-1), and sVEGFR-1 were determined by an enzyme-linked immunosorbent assay. The assays were performed as per the manufacturer’s instructions (Platinum ELISA kits; eBioscience, San Diego, CA). Urine albumin to creatinine ratios and high sensitivity C- reactive protein were determined using Tina-quant albumin Gen-2.ROCHE, Jaffe Gen.2-ROCHE, and Tina-quant C-reactive protein (Latex) kits in a COBAS INTEGRA 400/800 autoanalyzer. The urine albumin/creatinine ratio was calculated as milligrams per gram. The estimated glomerular filtration rate (eGFR) was calculated by using the following Modification of Diet in Renal Disease Study formula: glomerular filtration rate = 186 × [serum creatine] −1.154 × [age] −0.203 × [0.742 if patient is female] × [1.212 if patient is black].
Statistical analysis
Statistical analysis was performed with Prism Statistical software (version 5.0; GraphPad, San Diego, CA). Continuous variables were reported as mean ± SD unless otherwise specified. Comparisons of continuous variables between the groups were performed by Kruskal-Wallis-analysis of variance and Dunns post hoc test or Mann-Whitney U test where appropriate. Correlation analyses were performed by Spearman’s rank correlation analysis. Results were regarded as significant at P < .05.
Results
Comparison of study populations and cardiovascular risk factors
Demographic data of the study groups is shown in Table 1 . The mean age between the normal pregnancy and preeclampsia groups and previously normal pregnancy and previous preeclampsia groups were similar. The parity numbers were similar in all of the study groups. The pregnancy duration of both preeclampsia and normal pregnancy groups were similar. The time after the termination of pregnancy in the previous preeclampsia and the previous normal pregnancy groups were also similar. Body mass index (BMI) values were comparable between the normal pregnancy and preeclampsia groups. The BMI in the previous normal pregnancy group was lower than the previous preeclampsia group ( P < .05) ( Table 1 ).
| Characteristic | Normal pregnancy group (n = 21) | Preeclampsia group (n = 17) | Previous normal pregnancy group (n = 24) | Previous preeclampsia group (n = 16) |
|---|---|---|---|---|
| Age, y | 28.5 ± 3.5 | 28.9 ± 6.4 | 32.4 ± 5.6 | 35.5 ± 6.4 |
| Parity, n | 0.8 ± 0.7 | 0.9 ± 0.9 | 1.6 ± 0.8 | 2.3 ± 1.0 |
| Duration of pregnancy, wks | 33.3 ± 2.7 | 31.6 ± 4.3 | ||
| Time after the termination of pregnancy, mo a | 50.8 ± 52.3 26.0 (12–182) | 56.9 ± 48 38.5 (6–155) | ||
| BMI, kg/m 2 | 27.9 ± 3.0 | 29.5 ± 4.3 | 24.2 ± 3.7 b | 26.7 ± 3.8 |
| SBP, mm Hg | 110 ± 12 c | 159 ± 15 | 108 ± 13 c,d | 127 ± 19 c |
| DBP, mm Hg | 68 ± 7 c | 92 ± 11 | 70 ± 10 c,d | 83 ± 10 |
b P < .05 vs previous preeclampsia group
c P < .0001 vs preeclampsia group
d P < .0001 vs previous preeclampsia group. In previous preeclampsia group, 5 patients were on antihypertensive medication.
Five women in the previous preeclampsia group were on antihypertensive treatment. Systolic and diastolic blood pressures were higher in the preeclampsia group compared with the normal pregnancy group ( P < .0001) ( Table 1 ). Systolic and diastolic blood pressure values were higher in previous preeclampsia group than the previous normal pregnancy group in spite of the fact that 5 women in the previous preeclampsia group were on antihypertensive drugs ( P < .0001) ( Table 1 ).
Fasting blood glucose was similar in all of the groups. Total cholesterol, low-density lipoprotein-cholesterol, and triglyceride levels were higher in the preeclampsia group compared with the normal pregnancy group. Lipid profiles were similar in the previous preeclampsia and previous normal pregnancy groups ( P = NS). Uric acid levels were higher in the preeclampsia group, compared with the other 3 groups ( P < .0001, Table 2 ). The high-sensitivity C-reactive protein (hsCRP) values were higher in the preeclampsia group than the previous normal pregnancy group ( P < .0005) ( Table 2 ).
| Variable | Normal pregnancy group (n = 21) | Preeclampsia group (n = 17) | Previous normal pregnancy group (n = 24) | Previous preeclampsia group (n = 16) |
|---|---|---|---|---|
| Glucose, mg/dL | 76 ± 9 | 86 ± 22 | 82 ± 9 | 89 ± 16 |
| Cholesterol, mg/dlL | 226 ± 55 a | 272 ± 52 | 177 ± 33 b | 184 ± 35 c |
| LDL-cholesterol, mg/dL | 131 ± 40 a | 160 ± 44 | 98 ± 26 b | 107 ± 31 c |
| Triglyceride, mg/dL | 192 ± 113 a | 290 ± 106 | 99 ± 66 | 126 ± 73 c,d |
| HDL- cholesterol, mg/dL | 57 ± 13 | 53 ± 17 | 60 ± 15 | 51 ± 11 |
| Uric acid, mg/dL | 3.4 ± 0.8 c | 6.2 ± 1.7 | 3.7 ± 0.7 c | 4.0 ± 1.0 c |
| hsCRP, mg/L | 4.07 ± 2.9 | 21.9 ± 32.8 | 1.89 ± 2.6 c | 3.93 ± 4.7 |
a P < .01 vs preeclampsia group
b P < .01 vs normal pregnancy group
c P < .001 vs preeclampsia group
Number of CECs in the study populations
The CEC number was significantly higher in the preeclampsia group (9.9 ± 7.9 cells/mL) compared with the other 3 groups ( P < .0005, Table 3 ). The CEC number was similar between the previously preeclamptic and the previously normal pregnancy groups ( Table 3 , markers of endothelial function and damage in the study populations). No relationship between the CEC number and age, BMI, and systolic and diastolic blood pressure and adhesion molecule levels could be determined. In the preeclampsia group, there was a correlation between CEC number and hsCRP at a level that was not significant (r = 0.45; P = .064).
| Markers | Normal pregnancy group (n = 21) | Preeclampsia group (n = 17) | Previous normal pregnancy group (n = 24) | Previous preeclampsia group (n = 16) |
|---|---|---|---|---|
| CEC, per mL | 3.0 ± 4.1 a | 9.9 ± 7.9 | 3.4 ± 4.0 a | 2.4 ± 2.0 a |
| FMD, % | 10.1 ± 4.3 | b | 11.3 ± 4.6 | 10.2 ± 4.0 |
| eGFR, mL/min per 1.73 m 2 | 153 ± 27 a | 112 ± 28 | 112 ± 21 | 102 ± 20 |
| Ualb/creat, mg/g | 17.7 ± 18 c | 2167 ± 1793 | 9.4 ± 15.0 c | 51.08 ± 109 c,d |
a P < .0005 vs preeclampsia group
b Data are not present in preeclamptic group
c P < .0001 vs preeclampsia group
Systemic markers of endothelial function and vascular measurements
E-selectin and sVCAM-1 values were higher in the preeclampsia group than the normal pregnancy ( P < .0001), the previous preeclampsia and previous normal pregnancy groups ( P < .05) ( Table 4 , systemic markers of endothelial function and sVEGFR-1 in the study populations). The sICAM-1 levels were similar between the preeclampsia and the normal pregnancy groups. When the previous preeclampsia and previous normal pregnancy groups were compared, no significant difference was observed for the adhesion molecule levels.
