Endothelial damage and complement dysregulation in fetuses from pregnancies complicated by preeclampsia

Abstract

Introduction: Our objective was to evaluate the endothelial function profile and complement system in fetuses from preeclamptic pregnancies using ex vivo and in vitro approaches.

Material and methods: A total of 66 singleton pregnancies were prospectively recruited comprising 34 cases of preeclampsia and 32 normotensive pregnancies matched for baseline characteristics. In the ex vivo approach, soluble tumor necrosis factor-a receptor 1 (sTNFR1), vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), Von Willebrand factor (sVWF), terminal complement complex (sC5b-9), Factor H, complement component C3a and Factor Bb were analyzed in fetal cord blood samples. In the in vitro model, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), Von Willebrand factor (VWF), vascular endothelial cadherin (VE-Cadherin), endothelial nitric oxide synthase (eNOS), reactive oxygen species (ROS) and C5b-9 deposits were evaluated on endothelial cells in culture exposed to fetal sera or plasma.

Results: Increased sVCAM-1, sICAM- l and decreased Factor H and Factor Bb concentrations were detected in preeclampsia fetuses as compared to fetuses from normotensive mothers (509.4 ± 28 vs. 378.4 ± 34.3 ng/mL, 161.1 ± 11.9 vs. 114.8 ± 6.8, 199.6 ± 18.3 vs. 267.1 ± 15.4 ng/mL and 6.6 ± 0.7 vs. 10.3 ± 1.4 μg/mL respectively, p < 0.05) with similar results in sTNFR1, sVWF, sC5b-9 and C3a. Endothelial cells exposed to fetal sera from preeclampsia showed incremented expression of VCAM-1(38.1 ± 1.4% vs. 28.3 ± 1.6%, p < 0.01), ICAM-1 (12 ± 0.9% vs. 8.6 ± 0.6%, p < 0.05), VWF (43.5 ± 2.9% vs. 3.7 ± 0.3%, p < 0.05), and ROS (5 × 10¹³ ± 1 × 10¹² vs. 3.5 × 10¹³ ± 1.4 × 10¹², p < 0.01) with similar expression of VE-Cadherin and eNOS as compared to those exposed to control fetuses. While soluble C5b-9 was similar between the study groups (851.4 ± 177.5 vs. 751.4 ± 132.81 ng/mL, p > 0.05), significantly less C5b-9 deposits on endothelial cells were induced by fetal plasma from preeclamptic compared to normotensive mothers (fold change 0.08 ± 0.02 vs. 0.48 ± 0.13, p < 0.01).

Conclusions: High levels of endothelial adhesion molecules and oxidative stress products suggest endothelial damage and reduced in vitro deposition of C5b-9 indicates complement dysregulation in preeclampsia fetuses. More research is necessary to study the impact of preeclampsia on fetal vascular health and innate immunity.

Keywords: C5b‐9; complement system; endothelium; fetus; preeclampsia; vascular.

FIGURE 1

Comparison of circulating biomarkers between the study groups. Scattered boxplots show soluble endothelial damage and complement system biomarkers in the study populations. The line depicts the median and the whiskers point to the standard error of mean (SEM). sTNFR1, soluble tumor necrosis factor receptor 1; sVCAM‐1, soluble vascular cell adhesion molecule‐1; sICAM‐1, soluble intracellular adhesion molecule‐1; sVWF, soluble Von Willebrand Factor; sC5b‐9, soluble C5b‐9; Factor H, C3a and Factor Bb. *p < 0.05 and **p < 0.01 compared with normotensive pregnancies adjusted for gestational age at delivery and route of delivery.

FIGURE 2

Comparison of in vitro biomarkers in endothelial cells exposed to control and preeclampsia fetal sera. Left column: Scattered boxplots showing surface cover (SC) of endothelial damage biomarkers expression. The line depicts the median and the whiskers point to the standard error of mean (SEM). *p < 0.05 and **p < 0.01 compared with normotensive pregnancies. Right column: Representative microscopy images of VCAM‐1, ICAM‐1, VWF, VE‐Cadherin, and eNOS staining (green: VCAM‐1, VE‐Cadherin and eNOS; red: ICAM‐1 and VWF) on endothelial cells (4′,6‐diamidino‐2‐phenylindole–stained nuclei, blue) induced by exposure to fetal sera.

FIGURE 3

Reactive oxygen species (ROS) production by endothelial cells exposed to fetal sera. Left column: Results of ROS expressed as mean fluorescence intensity (MFI). The line depicts the median and the whiskers point to the standard error of mean (SEM). *p < 0.05 compared with normotensive pregnancies. Right column: Representative microscopy image of ROS staining (green) on endothelial cells induced by sera from preeclampsia fetuses and fetuses from normotensive mothers.

FIGURE 4

C5b‐9 deposits (dC5b‐9) on endothelial cells exposed to fetal plasma. Left column: C5b‐9 deposits expressed as fold increase of the surface cover with respect to the healthy internal control. The line depicts the median and the whiskers point to the standard error of mean (SEM). **p < 0.01 compared with normotensive pregnancies. Right column: Representative microscopy image of C5b‐9 deposition staining (red) on endothelial cells (4′,6‐diamidino‐2‐phenylindole–stained nuclei, blue) induced by activated plasma from preeclampsia fetuses and fetuses from normotensive mothers.