Impaired functional capacity of fetal endothelial cells in preeclampsia

Abstract

Objectives: Preeclampsia is one of the main contributers to maternal and fetal morbidity and mortality during pregnancy. A history of preeclampsia puts mother and offspring at an increased cardiovascular risk in later life. We hypothesized that at the time of birth functional impairments of fetal endothelial cells can be detected in pregnancies complicated by preeclampsia and that a therapeutic intervention using 1,25 (OH)2 vitamin D3 can reverse the adverse effects of preeclampsia on cell function.

Methods: Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords obtained from preeclamptic (N = 12) and uncomplicated pregnancies (N = 13, control). Placental villous tissue fragments from uncomplicated term pregnancies were incubated in explant culture for 48 h at 2% (hypoxia), 8% or 21% O2. Explant conditioned media (CM) was collected and pooled according to oxygen level. We compared the ability of preeclampsia vs. control HUVEC to migrate, proliferate, and form tubule-like networks in a Matrigel assay, in the presence/absence of CM and 1,25(OH)2 vitamin D3.

Results: HUVEC from preeclamptic pregnancies showed reduced migration (P = 0.04) and tubule formation (P = 0.04), but no change in proliferation (P = 0.16) compared to healthy pregnancies. Placental villous explant CM derived from 2% O2 incubations significantly reduced HUVEC migration, when compared to non-CM (P = 0.04). Vitamin D3 improved HUVEC function in neither of the groups. There was no significant difference in VEGF gene expression between healthy and preeclamptic pregnancies and no effect of Vitamin D3 on VEGF expression.

Conclusions: Reduced functional abilities of fetal endothelial cells from preeclamptic pregnancies suggests that disease pathways, possibly originating from the dysfunctional placenta, negatively impact fetal endothelium. The neutral effect of 1,25(OH)2 vitamin D3 contrasts with previous findings that vitamin D rescues the poor migration, proliferation and tubule formation exhibited by cord blood fetal endothelial progenitor cells from preeclamptic pregnancies. Further investigations to distinguish pathways by which offspring exposed to preeclampsia are at risk for cardiovascular disease are needed.

Fig 1. 25(OH) vitamin D concentrations (ng/ml) in the individual maternal serum samples prior delivery and umbilical cord blood after delivery.

*P < 0.05 vs. control. The ends of the whiskers represent the maximum and minimum measured values. Distribution was examined using Kolmogorov- Smirnov test. Continuous data were compared with unpaired t-test.

Fig 2. Building of tubule-like structures by uncomplicated pregnancy (control) and preeclampsia (PE) HUVEC, and the effect of 1,25(OH)₂ vitamin D₃, on capillary-tube formation by HUVEC in a Matrigel assay.

(A) Capillary-tube formation (average total tubule length per microscopic field) was analyzed after 6 h by visual microscopy at 2.5 magnification. Data are expressed as total tubule length in cm. *P < 0.05 vs. control. The ends of the whiskers represent the maximum and minimum measured values. Distribution was examined using Kolmogorov- Smirnov test. Continuous data were compared with Mann-Whitney test. (B) Representative photomicrographs of HUVEC after incubation in Matrigel. Scale bar represents 750 μm.

Fig 3. Effect of preeclampsia (PE), preeclampsia-like conditions and 1,25(OH)₂ vitamin D₃ on HUVEC migration.

(A) HUVEC of uncomplicated (control) and PE pregnancies were cultured in endothelial basal medium (EBM) and treated with or without 10 nM 1,25(OH)₂ vitamin D₃. The migration of PE HUVEC into the scratch wound assessed after incubation for 18 h was decreased compared to control. (B) "scratch wound" at 0 h and 8 h incubation is shown. Scale bar represents 750 μm. (C) Effect of villous explant conditioned medium (2% O₂, 8% O₂, 21% O₂ CM) and 1,25(OH)₂ vitamin D₃ on HUVEC migration with a reduction at 2% O₂. *P < 0.05 vs. untreated control. The ends of the whiskers represent the maximum and minimum measured values. Distribution was examined using Kolmogorov- Smirnov test. Continuous data were compared with unpaired t-test (A) or Wilcoxon-signed rank test (C).

Fig 4. Effect of pregnancy outcome and 1,25(OH)₂ vitamin D₃ on HUVEC population doubling time.

HUVEC of uncomplicated (control) and preeclamptic (PE) pregnancies were incubated in the presence and absence of 1,25(OH)₂ vitamin D₃ (10 nM). Cell numbers were counted and population doubling time calculated after 72 h. The ends of the whiskers represent the maximum and minimum measured values. Distribution was examined using Kolmogorov- Smirnov test. Continuous data were compared with Mann-Whitney test.

Fig 5. VEGF gene expression of HUVEC from uncomplicated (control) and preeclamptic (PE) pregnancies in the presence and absence of 1,25(OH)₂ vitamin D₃ (10 nM) in ug/ml.

For each treatment medium triplets were created and three RT-PCR runs were performed for each patient. The ends of the whiskers represent the maximum and minimum measured values. Distribution was examined using Kolmogorov- Smirnov test. Continuous data were compared with Mann-Whitney test.