Table 1.
Patient details for normal pregnant (n = 22) and preeclamptic women (n = 11).
Figure 1.
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.
Figure 2.
Four colour flow cytometric analysis of placental microvesicles.
Representative results for pSTBM analysis are shown in A(i-iv) and C(i-iv). Phenotyping of MV was carried out as follows: (Ai) Crude events >290 nm<1 µM were identified by FSC vs. SSC. (Aii) Aggregates were removed from the analysis using FSC-Height Vs FSC-Width, (Aiii) followed by SSC-Height Vs SSC-Width plots to resolve events >290 nm<1 µM. (Aiv) The total MV population was identified as bio-maleimide positive within this gate and this population was then used in subsequent analyses of MV associated ligands. pSTBM and mSTBM preparation purity was assessed using quadrant plots of NDOG2 (anti-placental alkaline phosphatase (PLAP) antibody) vs W6/32 (anti-MHC class I antibody) displaying the total MV population to identify syncytiotrophoblast derived MV (STBM) and other contaminating MV in (Bi, iii) normal pregnancy and (Bii, iv) preeclampsia (Bi, ii) mechanical and (Biii, iv) perfusion derived MV preparations respectively. The expression of Flt-1 and endoglin were investigated as follows: (Ci) Total MV and STBM populations were identified using bio-maleimide-Alexa 680 (Mal) staining and Mal with NDOG2-FITC double staining, respectively. Flt-1 or endoglin positive placental MV were identified by displaying the total MV population on either a (Cii) Flt-1-APC Vs NDOG2-FITC or (Ciii) endoglin-PE Vs NDOG2-FITC quadrant plot. Finally, (Civ) endoglin/Flt-1 double positive placental MV were identified by plotting Mal/NDOG2 double positive MV onto an endoglin-PE Vs Flt-1-APC quadrant plot. (D) Flow chart to describe the flow cytometric identification of syncytiotrophoblast derived microvesicles.
Figure 3.
Bar charts showing results of multi-colour flow cytometry analysis of (A) mechanically derived (mSTBM) and (B) perfusion derived (pSTBM) placental vesicle preparations.
(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.
Figure 4.
Western blotting analysis of placental vesicle preparations.
A) Representative immunoblot images and B) the corresponding densitometric analysis of placental alkaline phosphatase (PLAP), VEGF receptor-1 (Flt-1) and endoglin in syncytiotrophoblast microvesicles (STBM) prepared by i) mechanical disruption (mSTBM) and ii) placental perfusion (pSTBM) of normal and preeclampsia (PE) affected pregnancies. P values show the comparison between normal and PE derived pSTBM.
Figure 5.
Interaction of placental vesicle preparations with angiogenic factors.
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.
Figure 6.
Analysis of vesicle size distribution and exosome marker expression of SH-SY5Y neuroblastoma cell line and placental vesicle preparations.
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.