siRNA-mediated downregulation of caveolin-1 expression in BeWo cells.

<p><i>A</i>, BeWo cells were transfected with the indicated concentrations of caveolin-1(Cav-1) siRNA or a non-silencing control siRNA. After 48 h cells were lysed and levels of caveolin-1 and β-actin were assessed by immunoblotting. <i>B</i>, densitometric analysis of immunoblots assessed for caveolin-1 expression and normalised to β-actin expression, in cells transfected for 48 h with 50 nM Cav-1 siRNA or non-silencing control siRNA. Results are presented as mean ± SEM for three separate experiments, *p<0.001 compared with control transfected cells (determined by ANOVA).</p

sEndoglin levels in maternal plasma during labour (A, B) and Caesarean section (C,D).

<p>Median and ranges for sEndoglin ng/ml. In normal pregnancy labour (A) the levels declined by 24 hr (a***p<0.001, pre-labour vs 24 hr, b**p<0.01-full dilatation vs 24 hr, and placental delivery vs 24 hr). In pre-eclampsia labour (B) a similar decline by 24 h was noted (a*p<0.05, pre-labour vs 24 hr, full dilatation vs 24 hr, and placental delivery vs 24 hr). At Caesarean section (C,D) a significant decline in levels of sEndoglin by 24 hr was noted with placental delivery in both normal pregnancy and pre-eclampsia (a**p<0.01).</p

Interaction of placental vesicle preparations with angiogenic factors.

<p>Binding of Ai) VEGF₁₂₁, VEGF₁₆₅ and PlGF and Aii) TGFβ to increasing amounts of pooled mSTBM from 13 normal placentas. B) Representative fluorescence micrographs and C) the corresponding percentage coverage data for human umbilical vein endothelial cell monolayers treated with VEGF₁₂₁, VEGF₁₆₅, PlGF and TGFβ (100 ng/mL) in the presence and absence of normal mSTBM (75 µg/mL, pool of 13 preparations). Median values are indicated on the graphs. **P<0.001 compared with untreated control wells.</p

Inhibin A levels (n = 10) in maternal plasma during labour and Caesarean section.

<p>Median and ranges for inhibin A levels pg/ml in labour (A, B). In normal pregnancy labour (A) there was a significant decline in inhibin A levels by 24 h (a**p<0.01-prelabour vs 24 hr, full dilatation vs 24 hr, and b* p<0.05-placental delivery vs 24 hr. A similar decline was present in PE labour (B) (a*p<0.05, a-pre-labour vs 24 hr, b **p<0.01-placental delivery vs 24 hr), but the increase noted between pre-labour and full dilatation in pre-eclamptic women was not significant. At Caesarean section (C,D) a significant decline in levels of inhibin A by 24 hr was noted with placental delivery. (NP and PE a**p<0.01 placental delivery vs 24 hr).</p

Activin A levels (median (ranges) ng/ml) in maternal plasma during labour (A,B) and Caesarean section (C,D) in normal pregnancy (n = 10) and women with pre-eclampsia (n = 10).

<p>In normal pregnancy labour (A) there was a significant decline in activin A levels by 24 hr (a**p<0.01, pre-labour vs 24 hr, b ***p<0.001-full dilatation vs 24 hr, and c***p<0.001-placental delivery vs 24 hr. In pre-eclampsia labour (B) there was a significant increase in levels of activin A, (a*p<0.05 pre-labour vs full dilatation) and a significant decline postpartum (a*p<0.05 pre-labour vs 24 hr, b***p<0.001-full dilatation vs 24 hr, and c**p<0.01-placental delivery vs 24 hr). At Caesarean section a significant decline in levels of activin A by 24 hr was noted with placental delivery in normal pregnancy and pre-eclampsia (C,D). (NP a*p<0.05 and PE, a ***p<0.001 placental delivery vs 24 hr).</p

Elective Caesarean Section study.

<p>Elective Caesarean Section study.</p

Labour study.

<p>Labour study.</p

LSRII Flow Cytometer set up.

<p>A) SSC & FSC voltages were adjusted to visualise 290 nm and 1 µm microspheres to establish the microvesicle analysis gate. B) Trucount beads were analysed for two minutes demonstrating large numbers of background particles present in the Trucount tube. C) Two minute analysis of 0.1 µm filtered PBS showing the low number of background events present. D) FSC vs. SSC density plots with ≤1 µm gate of representative i) mSTBM and ii) pSTBM preparations and iii) barchart showing a significantly lower percentage of events ≤1 µm in both normal and PE mSTBM compared to pSTBM. *P<0.05 and **P<0.01.</p

Analysis of vesicle size distribution and exosome marker expression of SH-SY5Y neuroblastoma cell line and placental vesicle preparations.

<p>Nanosight Tracking Analysis size distribution profiles for (A) a representative SH-SY5Y neuroblastoma cell line exosome preparation, (B) mechanically derived syncytiotrophoblast microvesicle preparations and (C) placental perfusion (pSTBM) from normal (norm) and preeclampsia. (D) Representative immunoblot images and (E) the corresponding densitometric analysis of the exosome markers Lamp I, Alix, CD63 and CD9 in i) mSTBM and ii) pSTBM. Densitometric values were normalized using actin as a loading control.</p

Bar charts showing results of multi-colour flow cytometry analysis of (A) mechanically derived (mSTBM) and (B) perfusion derived (pSTBM) placental vesicle preparations.

<p>(i) Percentage positive events and (ii) mean fluorescence intensity of placental alkaline phosphatase (PLAP), Flt-1 (vascular endothelial cell growth factor receptor-1) and endoglin stained total microvesicle (MV) population (for PLAP) and STB derived MV (for Flt-1 and endoglin). p values within a barchart are a comparison between PE and normal whereas * denotes a comparison between mSTBM and pSTBM. *P<0.05 compared with normal mSTBM.</p