Objective
We sought to determine if endothelial microparticles (EMPs), markers of endothelial damage, are associated with soluble fms-like tyrosine kinase 1 (sFlt1), soluble endoglin, and placental growth factor (PlGF) in women with preeclampsia.
Study Design
A prospective cohort study was conducted on 20 preeclamptic women and 20 controls. EMPs by flow cytometry, sFlt1, soluble endoglin, and PlGF were measured at time of enrollment, 48-hours postpartum, and 1-week postpartum.
Results
Preeclamptic CD31 + /42 − , CD62E + , and CD105 + EMP levels were significantly elevated in preeclamptics vs controls at time of enrollment. The sFlt1:PlGF ratio was correlated with CD31 + /42 − and CD105 + EMPs (r = 0.69 and r = 0.51, respectively) in preeclampsia. Levels of CD31 + /42 − EMPs remained elevated 1-week postpartum ( P = .026).
Conclusion
EMPs are elevated in preeclampsia. The correlation of EMPs and the sFlt1:PlGF ratio suggests that antiangiogenesis is related to apoptosis of the endothelia. Endothelial damage persists 1 week after delivery.
Preeclampsia is a hypertensive disease of pregnancy that manifests as blood pressure elevation with new-onset proteinuria occurring >20th week of gestation. This disease complicates up to 7% of pregnancies, accounting for significant maternal and neonatal morbidity and mortality. The pathophysiologic insult occurring in preeclampsia is thought to be diffuse endothelial cell dysfunction and injury. This endothelial insult, and thereby the clinical manifestations of preeclampsia, appear to be related to the release of circulating factors from the placenta and their effects on the maternal vasculature. Soluble fms-like tyrosine kinase 1 (sFlt1) competitively binds to placental growth factor (PlGF) and vascular endothelial growth factor (VEGF), preventing their role in endothelial preservation. Soluble endoglin (sEnd) also serves as an antiangiogenic protein and has been shown to prevent capillary tube formation and increase vascular permeability by inhibition of the transforming growth factor beta 1 signaling pathway. The placenta produces sFlt1 and sEnd during normal pregnancy, but significantly higher amounts are produced from the hypoxic placenta in those pregnancies affected by preeclampsia. The presence of these proteins and their effect on PlGF and VEGF create an angiogenically imbalanced vascular environment that contributes to the endothelial insult occurring in preeclampsia. The role of the placenta in this process is further evidenced by the fact that the only true “cure” for preeclampsia is delivery, with symptoms resolving shortly after delivery. Typically sFlt1 and sEnd decrease rapidly after delivery; however the clearance rate is much longer in women with preeclampsia.
Recent studies provide evidence that identification of circulating endothelial microparticles (EMPs) allows direct assessment of endothelial damage. EMPs are submicroscopic membranous particles that are shed from the endothelial cell wall upon endothelial disturbance and can be identified within the plasma by fluorescent antibody labeling of endothelial cell adhesion molecules retained on the surface of the liberated EMPs. Quantification via flow cytometry provides a direct measurement of endothelial damage. Moreover, phenotypic variability of the EMPs identified by differential expression of specific cell surface markers may suggest the underlying endothelial insult taking place. EMP elevations have been documented in disease states with known endothelial insult, including preeclampsia. The ability to directly assess endothelial insult allows the relationship between the maternal endothelial environment and the circulating antiangiogenic factors in preeclampsia to be explored.
This study had 2 main objectives designed to examine both antepartum and postpartum endothelial function. First, we evaluated EMPs in the antepartum period as objective markers of endothelial damage in women with preeclampsia and determined if levels of liberated EMPs are related to the angiogenic and antiangiogenic proteins associated with preeclampsia. Second, we evaluated EMP levels in the postpartum period to determine whether endothelial damage persists after delivery.
Materials and Methods
Study subjects
A prospective, case-control study was conducted on 20 preeclamptic women and 20 healthy pregnant women recruited from Parkland Memorial Hospital, Dallas, TX, and its affiliated prenatal care clinics from February 2009 through June 2010. Eligibility for enrollment included women with singleton gestations ≥36 weeks. For study subjects, the diagnosis of preeclampsia was defined as blood pressure measurement ≥140/90 mm Hg on 2 occasions within 6 hours in a previously normotensive woman and ≥2+ proteinuria in a catheterized urine specimen. These patients were recruited from the Labor and Delivery Unit at Parkland Memorial Hospital immediately following the diagnosis of preeclampsia and prior to the administration of magnesium sulfate. Control subjects were healthy pregnant women with singleton gestations, a normal blood pressure, and no proteinuria. These patients were recruited during their routine prenatal care visit and were matched to preeclamptic subjects for maternal age (±2 years), gestational age at enrollment (±1 week), and parity. Exclusion criteria for all participants included: preexisting hypertension, proteinuria <24 weeks’ gestation, pregestational or gestational diabetes, renal or hepatic disease, or known fetal anomalies. Informed consent was obtained from all subjects, according to protocols approved by the institutional review boards of University of Texas Southwestern and Parkland Memorial Hospital.
Collection of blood samples
Blood was collected at time of diagnosis of preeclampsia or study enrollment, 48-hours postpartum (±12 hours), and 1-week postpartum (±1 day). A manual blood pressure measurement was taken prior to venipuncture. Blood samples were collected without tourniquet in sterile 5-mL sodium citrate tubes for microparticle analysis and 5-mL EDTA tubes for enzyme-linked immunosorbent assay analysis (BD Vacutainer Systems, Franklin Lakes, NJ).
Microparticle analysis
Samples for microparticle analysis were assayed within 2 hours of venipuncture to avoid contamination with microparticles released ex vivo. To avoid freeze-thaw cycles, the microparticle samples were not frozen as we found similar to previous reports that freezing results in variability in microparticle measurements. Centrifugation was carried out to obtain plasma depleted of platelets, cells known to release platelet–specific microparticles. The samples were centrifuged 10 minutes at 0.1 g to prepare platelet-rich plasma, which was centrifuged 6 minutes at 0.2 g, then 1 minute at 3.3 g to obtain platelet-poor plasma (PPP). Aliquots of 50 μL of PPP were transferred into individual microcentrifuge tubes for antibody staining. For measurement of CD31 + /42 − EMP, the PPP was incubated with 1 μL of flourescein isothiocyanate (FITC)-labeled anti-CD42 antibody and 2 μL of phycoerythrin (PE)-labeled anti-CD31 antibody (catalog nos. 555472 and 555446, respectively; BD Pharmingen, San Jose, CA). Double labeling with anti-CD42b was for the exclusion of CD31 + microparticles of platelet origin. CD31 + /CD45 + particles account for a negligible percentage of all CD31 + microparticles, which would represent a subpopulation of leukocyte microparticles that are not considered to significantly alter EMP counts. For measurement of CD62E + and CD105 + EMP, the PPP was incubated with 0.5 μL of PE-labeled CD62E antibody or 0.5 μL of PE-labeled CD105 antibody (catalog nos. 551144 and 560839, respectively; BD Pharmingen). Antibody incubation time was 15 minutes, shielded from light with gentle orbital shaking. To facilitate removal of excess antibody, 500 μL of filtered phosphate buffered saline was added to each sample following incubation. The samples were then centrifuged at 20,000 g for 10 minutes at 20°C. Using a transfer pipette, 450 μL of supernatant was gently removed. The sedimented microparticles were resuspended in 500 μL of filtered phosphate buffered saline and analyzed by flow cytometry.
Two-color flow cytometry was performed on a FACSCalibur flow cytometer equipped with CellQuest software (BD Biosciences). Thresholds for forward scatter (FSC) and side scatter were set to 0. FSC, side scatter, and fluorescence channels were set at logarithmic gain. We used 1-μm beads to provide the standard for FSC gate determination of microparticle size (catalog no. F-13838; Invitrogen, Grand Island, NY). Microparticles were then identified on the basis of their size, density, and fluorescence. Analysis of each sample was performed for 45 seconds at medium flow rate. Three consecutive analyses were performed for each sample and the median event count deemed the final count. The absolute concentration of circulating microparticles was calculated using calibrator beads with known concentration added into the sample immediately prior to flow cytoanalysis (catalog no. NT20N/9207; Bangs Laboratories, Grand Island, NY). The final microparticle number was expressed as count/μL.
Protein analysis
The serum samples utilized for measurement of sFlt1, sEnd, and PlGF were centrifuged and stored at –70°C. The concentrations of sFlt1, sEnd, and PlGF were determined by using specific enzyme-linked immunosorbent assay following the manufacturer’s instructions (R&D Systems Inc, Minneapolis, MN). All samples were examined in duplicate and mean values of individual sera were utilized for statistical analysis. The minimum detectable doses in the assays for sFlt1, sEnd, and PlGF were 3.5, 7, and 7 pg/mL, respectively. The intraassay and interassay coefficients of variation were 3.2% and 5.5%, respectively, for sFlt1; 3.0% and 6.3%, respectively, for sEnd; and 5.6% and 10.9%, respectively, for PlGF.
Statistical analysis
A sample size of 20 study subjects per group was selected to allow with 80% power a detection of a difference of 5000 EMP/μL between preeclampsia and controls assuming SD of 5000 EMP/μL. Assumptions are based upon previously published studies. Control patients were matched to cases by maternal age, gestational age at enrollment, and parity as stated earlier. Demographic data are expressed as mean + SD, analyzed using the Student t test or expressed as frequency (percent), analyzed using the Pearson χ 2 test. In the case of birthweight, analysis of covariance was used to adjust the measure of association by gestational age. EMP data are not assumed to be statistically normal an assumption examined using the Shapiro-Wilk test. Consequently, 2-group comparisons are made using the Wilcoxon rank sum test. Correlations were estimated using the Spearman correlation. Confidence intervals of correlations are estimated using the Fisher transformation and the hypothesis of equality of correlation between groups uses the Student t test again through the Fisher transformation. For the longitudinal results, data were transformed to statistical normality using the logarithmic transformation. Following transformation, the Shapiro-Wilk test was used to substantiate that the transformation did indeed result in normal data. As some subjects did not have data in all 3 of the time epochs, the repeated measure of time is a random effect. Consequently, the analysis for these longitudinal data is a random effects model with subject grouping (preeclampsia or control) as a fixed effect and the repeated measure for time as a random effect at the fixed time points of: enrollment, 2-days postpartum, and 1-week postpartum. Consequently, the study design is a repeated measures analysis of variance with the repeated measure as a random effect. If the overall statistic of the random effects model is significant for the fixed effect of subject grouping, then individual contrasts are examined for the cross-sectional comparisons at each time point. All analysis is conducted in the transformed logarithmic domain. P levels < .05 are assumed to be significant. Due to variations in disease severity, EMP and protein levels were reported as a median value (interquartile range). Statistical analyses were performed using software (SAS, version 9.2; SAS Institute, Cary, NC).
Results
The demographic and clinical characteristics of the study subjects are presented in the Table . There were no significant differences in maternal age, race, parity, gestational age at enrollment, or smoking. Women with preeclampsia had a significantly higher prepregnancy body mass index than control subjects ( P < .015). Women with preeclampsia had a higher mean arterial blood pressure than control subjects. The mode of delivery was similar between the 2 groups. The birthweight was significantly lower in the preeclamptic group, but nonsignificant after adjusting for gestational age at the time of delivery.
| Demographic | Preeclampsia (n = 20) | Control (n = 20) | P value |
|---|---|---|---|
| Maternal age, y | 25.1 ± 7.0 | 25.1 ± 6.7 | NS |
| Race | |||
| African American | 1 (5) | 1 (5) | NS |
| Hispanic | 19 (95) | 19 (95) | NS |
| Parity | |||
| Nulliparous | 12 (60) | 12 (60) | NS |
| Multiparous | 8 (40) | 8 (40) | NS |
| Prepregnancy BMI | 28.5 ± 5 | 23.9 ± 5 | .015 |
| Smoking | 1 (5) | 1 (5) | NS |
| Enrollment GA | 38.6 ± 1.8 | 38.4 ± 1.4 | NS |
| MAP, mm Hg | |||
| Enrollment | 115 ± 8 | 79 ± 7 | < .001 |
| 2 d PP (n = 14) | 105 ± 9 | 90 ± 9 | < .001 |
| 1 wk PP (n = 15) | 101 ± 12 | 87 ± 10 | < .001 |
| Mode of delivery | |||
| Vaginal | 14 (70) | 13 (65) | NS |
| Cesarean | 6 (30) | 7 (35) | NS |
| Birthweight, g | 2998 ± 485 | 3543 ± 494 | < .001 a |
a Nonsignificant when adjusted for gestational age at delivery.
Antepartum period
The levels of all EMPs measured were elevated in women with preeclampsia ( Figure 1 ). CD31 + /42 − EMPs were significantly higher in preeclamptic subjects as compared to control subjects (18,552 [12,456–21,624] vs 7606 [5755–13,105] counts/μL, P < .001). CD62E + EMPs were increased in women with preeclampsia over control patients (1231 [884–1661] vs 739 [452–1023] counts/μL, P < .001). Levels of CD105 + EMPs were also significantly higher in women with preeclampsia (1932 [1210–2417] vs 737 [553–1052] counts/μL, P = .003).
