Maternal endothelial progenitor colony-forming units with macrophage characteristics are reduced in preeclampsia

Abstract

Background: Endothelial progenitor cells (EPCs) provide paracrine support to the vascular endothelium and may also replace damaged or senescent endothelial cells. Low numbers of endothelial progenitor colony-forming units (CFU-ECs) in culture are a predictive biomarker of vascular disease. We hypothesized that the number of CFU-ECs derived from maternal blood are decreased in women with preeclampsia compared to normal pregnancy.

Methods: Primigravid women with singleton normal (n = 12) or preeclamptic (n = 12) pregnancies were studied during the third trimester. The culture assay was performed using a pre-plating step to eliminate mature endothelial cells and nonprogenitor cells; colonies per well were counted and further characterized.

Results: Colony numbers were fourfold lower on average in preeclampsia compared to control samples (P < 0.005). A majority of the cells comprising individual colonies were positive for both endothelial (Ulex europaeus lectin staining and acetylated low-density lipoprotein (LDL) uptake) and monocyte/macrophage (CD45, CD14, CD115) characteristics. The SRY gene was detected in CFU-ECs derived from umbilical cord blood samples from male fetuses but not in CFU-ECs from peripheral blood of mothers with male fetuses. Maternal plasma concentrations of the antiangiogenic factor, soluble fms-like tyrosine kinase-1 (sFlt-1) were elevated (P < 0.0001) whereas placental growth factor (PlGF) was reduced (P < 0.01) in women with preeclampsia, but these factors did not correlate with CFU-EC counts.

Conclusions: CFU-ECs derived from culture of peripheral blood mononuclear cells, a correlate of cardiovascular risk in nonpregnancy populations, are rarified in women with preeclampsia compared to normal pregnancy. PCR analysis is consistent with a maternal origin of these cells.

Figure 1

Scatterplot of colony-forming unit counts during the third trimester. Each symbol corresponds to average values per culture well from a different individual.

Figure 2

No evidence of fetal endothelial progenitor cells (EPCs) in maternal blood by genomic PCR for SRY gene. Reactions contained 0.5 μg DNA- and SRY-specific primers. PCR product (242 bp) was electrophoresed on 2% agarose. (a) Lane 1, 100 bp DNA ladder; lane 2, EPCs from adult male; lanes 3, 5, 7 and 9, EPCs from maternal blood; lanes 4 and 8, umbilical cord blood EPCs from male fetus (positive control); lanes 6 and 10: umbilical cord blood EPCs from female fetus. (b) The 133 bp region of rhodopsin gene (RDP) was amplified as a control for loading.

Figure 3

Characteristics of colony-forming units (CFUs) in culture derived from maternal blood samples (original magnification ×100), representative of three patients with normal pregnancy and three with preeclampsia. (a) Brightfield image of CFU in culture. (b and c) Same CFU as a, showing accumulation of Dil-acLDL (red) and FITC-conjugated Ulex lectin (green), respectively. (d) Brightfield image of CFU. (e) Same CFU as d, showing overlay of DAPI nuclear staining (blue) and CD45 (red). (f) Control for e and g, showing overlay of DAPI staining and absence of immunoreactivity to mouse antihuman IgG isotype control followed by goat antimouse Alexa Fluor 568 secondary antibody. (g) Overlay of DAPI and CD14 (red). (h) Overlay of DAPI and CD115 (red). (i) Control for h, showing overlay of DAPI staining and absence of immunoreactivity to rabbit antihuman IgG G1 plus goat antirabbit Alexa Fluor 568 secondary antibody.