The role of VEGF-A165b in trophoblast survival.

BACKGROUND: Pre-eclampsia remains a dominant cause of maternal and fetal mortality in developed countries. In a previous prospective study we identified a fall in the VEGF-A isoform VEGF-A165b in the plasma of patients in the first trimester to be a predictor of later pre-eclampsia. VEGF-A165b has been shown to have potent cytoprotective properties in many cell types. We therefore tested the hypothesis that VEGF-A165b may be cytoprotective for placental trophoblasts. METHODS: We used an immortalised first trimester trophoblast cell line exposed to chemical toxicity, and physiological (<2% O2) and atmospheric oxygen (21% O2) in the presence or absence of VEGF-A165b, angiogenic VEGF-A165a, a non-specific anti-VEGF-A blocking antibody (bevacizumab), or a specific anti-VEGF-A165b antibody. Cell viability and cytotoxicity were measured by trypan blue and LDH assay respectively. RESULTS: Under high (21%) levels of oxygen, trophoblast viability was increased, and cytotoxicity reduced by exogenous recombinant VEGF-A165b (p < 0.05, n = 10) or VEGF-A165a. The cytoprotective effect was not seen under lower (<2%) oxygen conditions, where VEGF-A165b was upregulated. However inhibition of VEGF-A with blocking antibodies (bevacizumab or anti-VEGF-A165b) had marked cytotoxic effects under low oxygen conditions presumably through the blockade of autocrine survival pathways. CONCLUSIONS: These results show that when trophoblasts are exposed to lower oxygen tensions (as they are early in the 1st trimester) endogenous VEGF-A165b contributes to their survival through an autocrine pathway. In contrast in high oxygen conditions exogenous VEGF-A isoforms have a greater effect on trophoblast survival.

Failure to up-regulate VEGF165b in maternal plasma is a first trimester predictive marker for pre-eclampsia.

Pre-eclampsia is a pregnancy-related condition characterized by hypertension, proteinuria and endothelial dysfunction. VEGF(165)b, formed by alternative splicing of VEGF (vascular endothelial growth factor) pre-mRNA, inhibits VEGF(165)-mediated vasodilation and angiogenesis, but has not been quantified in pregnancy. ELISAs were used to measure means+/-S.E.M. plasma VEGF(165)b, sEng (soluble endoglin) and sFlt-1 (soluble fms-like tyrosine kinase-1). At 12 weeks of gestation, the plasma VEGF(165)b concentration was significantly up-regulated in plasma from women who maintained normal blood pressure throughout their pregnancy (normotensive group, 4.90+/-1.6 ng/ml; P<0.01, as determined using a Mann-Whitney U test) compared with non-pregnant women (0.40+/-0.22 ng/ml). In contrast, in patients who later developed pre-eclampsia, VEGF(165)b levels were lower than in the normotensive group (0.467+/-0.209 ng/ml), but were no greater than non-pregnant women. At term, plasma VEGF(165)b concentrations were greater than normal in both pre-eclamptic (3.75+/-2.24 ng/ml) and normotensive (10.58 ng/ml+/-3.74 ng/ml; P>0.1 compared with pre-eclampsia) pregnancies. Patients with a lower than median plasma VEGF(165)b at 12 weeks had elevated sFlt-1 and sEng pre-delivery. Concentrations of sFlt-1 (1.20+/-0.07 and 1.27+/-0.18 ng/ml) and sEng (4.4+/-0.18 and 4.1+/-0.5 ng/ml) were similar at 12 weeks of gestation in the normotensive and pre-eclamptic groups respectively. Plasma VEGF(165)b levels were elevated in pregnancy, but this increase is delayed in women that subsequently develop pre-eclampsia. In conclusion, low VEGF(165)b may therefore be a clinically useful first trimester plasma marker for increased risk of pre-eclampsia.

Imaging the placental glycocalyx with transmission electron microscopy.

There is a significant glycocalyx present at the maternal-fetal interface of the human placenta, with increasing evidence to suggest it has an important role in placental function. Glycocalyx is adversely affected by traditional tissue processing and fixation techniques. Using transmission electron microscopy, we present methodologies for reliably imaging and measuring glycocalyx of both the syncytiotrophoblast and fetal capillary endothelium in term healthy placentae. These techniques can be used to study the role of the placental glycocalyx in both health and disease, including pre-eclampsia.