Daniel A. Plymire

Novel Soluble Flt-1 Isoforms in Plasma and Cultured Placental Explants from Normotensive Pregnant and Preeclamptic Women

Pregnant women who develop preeclampsia exhibit higher circulating levels of the soluble VEGF receptor-1 (sFlt-1). Recent findings suggest that soluble Flt-1 may contribute to the pathogenesis of preeclampsia by binding and neutralizing vascular endothelial growth factors (VEGF) and placental growth factor (PlGF). Existing literature identifies sFlt-1 as a 100 kDa glycoprotein, a product of an mRNA splice variant. We hypothesized that sFlt-1 expression may be more complex with multiple variants of sFlt-1 as well as multiple sources during normal pregnancy and preeclampsia. Using a combination of affinity purification of sFlt-1 by heparin-agarose and epitope specific antibodies, we performed Western blot analysis with epitope specific antibodies for sFlt-1. Plasma of preeclamptic women exhibits significantly higher amounts of a novel 145 kDa variant of sFlt-1, along with the 100 kDa isoform. We identified sFlt-1 variants in the conditioned medium from placental explant cultures that are hypoxia responsive with varying sizes, including 185, 145,100 and 60 kDa forms, as well as antigenicity. The 145 kDa was similar in antigenicity to the 100 kDa found in plasma whereas the 185 and 60 kDa sFlt-1 demonstrated different epitopes. Deglycosylation studies also confirm that there are multiple sFlt-1 polypeptides. Co-immunoprecipitation with VEGF suggests that these different sFlt isoforms can bind VEGF and therefore, may be of functional importance. Finally, comparison of sFlt-1 in the conditioned medium obtained from cultured cytotrophoblasts, peripheral blood mononuclear cells (PBMCs) and human uterine microvascular cells (HUtMVECs) exhibit mainly the100 kDa sFlt-1. Collectively these data suggest the presence of multiple isoforms of sFlt-1 in the circulation of women with preeclampsia as well as in uncomplicated pregnancies and the possibility of multiple sources. Placental hypoxia may contribute to sFlt-1 over expression but other regulatory mechanisms cannot be ruled out.

Maternal Circulating CD34+VEGFR-2+ and CD133+ VEGFR-2+ Progenitor Cells Increase During Normal Pregnancy but Are Reduced in Women With Preeclampsia

Circulating endothelial progenitor cells (EPCs) may contribute to vascular endothelial cell homeostasis, and low levels of these cells are predictive of cardiovascular disease. We hypothesized that circulating EPCs increase in number during uncomplicated pregnancy but are reduced in women with preeclampsia. Peripheral blood was obtained from pregnant women and from nulligravidas in cross-sectional design. Cells expressing CD34 or CD133, in combination with vascular endothelial growth factor receptor-2 (VEGFR-2), were enumerated by flow cytometry. Both CD34+VEGFR-2+ (doubly positive) and CD133+VEGFR-2 + cells were significantly increased during the second and third trimesters of uncomplicated pregnancy compared to the first trimester. First trimester and nulligravida groups did not differ. Endothelial progenitor cells, quantified by flow cytometry or by circulating angiogenic cell (CAC) culture assay, were significantly reduced in women with preeclampsia compared to third trimester controls. Circulating EPCs appear to increase during normal pregnancy, and comparatively reduced numbers of these cells exist during preeclampsia.

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

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.