Knockdown of VASH2 inhibited the forskolin-induced fusion of BeWo cells.

<p>A: BeWo cells with or without siRNA treatment were stimulated with FK, and cell fusion was observed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104728#s2" target="_blank">Materials and Methods</a>. Bar = 100 µm. Arrows indicate fused cells with multiple nuclei. B: Expression of human VASH2 was quantified (N = 3). *P<0.01, NS; not significant. C: Cell fusion was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104728#s2" target="_blank">Materials and Methods</a> (N = 2, 10 fields each). *P<0.01. D–F: Expression of Gcm-1, Syn-2, and Syn-1 in BeWo cells with each treatment (N = 3) was determined by qRT-PCR. NS; not significant.</p

Localization of VASH1 and VASH2 in human placenta.

<p>Immunohistochemical analysis for the localization VASH1 (A) and VASH2 (B) in the human placenta was performed. Arrowheads indicate VASH1 vessels (A). Bar = 100 µm.</p

The overexpression of VASH2 augmented the fusion of BeWo cells.

<p>A: BeWo cells were infected with adenovirus vectors. Expression of human VASH2 was quantified by qRT-PCR (N = 3). B: Cell fusion was observed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104728#s2" target="_blank">Materials and Methods</a>. Bar = 100 µm. Arrowheads indicate a fused cell with multiple nuclei. Cell fusion was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104728#s2" target="_blank">Materials and Methods</a> (N = 3, 5 fields each). #</p

Course of pregnancy in WT, <i>Vash1^(−/−)</i> and <i>Vash2^(−/−)</i> mice.

<p>A: Comparison of maternal weights of WT (N = 30), <i>Vash1^(−/−)</i> (N = 45), and <i>Vash2^(−/−)</i> (N = 20) mice. *P<0.01. B: Comparison of number of neonates per WT (N = 32), <i>Vash1^(−/−)</i> (N = 45), and <i>Vash2^(−/−)</i> (N = 20) dams. C: Blood pressure of WT (N = 16), <i>Vash1^(−/−)</i> (N = 15), and <i>Vash2^(−/−)</i> (N = 7) dams measured at 0, 4.5, 8.5,10.5, 12.5, 14.5, 16.5, and 18.5 dpc. #P<0.05. D: Wet weight of WT (N = 37), <i>Vash1^(−/−)</i> (N = 37), and <i>Vash2^(−/−)</i> (N = 19) placentas. *P<0.01.</p

Vascularization of placenta in WT, <i>Vash1^(−/−)</i>, and <i>Vash2^(−/−)</i> mice.

<p>Upper panels show vascular morphogenesis. The triple staining with tomato lectin (green), anti-CD31 (blue), and anti-type IV collagen (red) was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104728#s2" target="_blank">Materials and Methods</a>. Tomato lectin identified the maternal blood vessels; and CD31-positive structures, the fetal blood vessels. The presence of type IV collagen indicated the basement membrane. Bar = 50 µm. Lower graph on the left show the fetal vascular area, and that on the right shows the maternal vascular area determined for WT (N = 5), <i>Vash1^(−/−)</i> (N = 3), and <i>Vash2^(−/−)</i> (N = 3) placentas. Ten 400× fields per placenta were used for quantification. *P<0.01.</p

Serum levels of VEGF, sVEGFR1, and PlGF in WT, <i>Vash1^(−/−)</i>, and <i>Vash2^(−/−)</i> dams.

<p>A: Serum levels of VEGF-A at 12.5, 16.5, and 18.5 dpc. The respective numbers of WT dams at these time points were 6, 3 and 4; of <i>Vash1^(−/−)</i> ones, 7, 10 and 22; and of <i>Vash2^(−/−)</i> dams, 4, 4 and 8. ^#P<0.05, *P<0.01. B: Serum levels of sVEGFR1 at 12.5, 16.5, and 18.5 dpc. The respective numbers of WT dams at these time points were 5, 4, and 5; of <i>Vash1^(−/−)</i> ones, 6, 8 and 17; and of <i>Vash2^(−/−)</i> dams, 4, 4, and 5. C: Serum levels of PlGF at 12.5, 16.5, and 18.5 dpc. Respective numbers of WT dams at these stages were 4, 2 and 2; of <i>Vash1^(−/−)</i> ones, 5, 5, and 5; and of <i>Vash2^(−/−)</i> dams, 3, 3, and 4.</p

Labyrinth and syncytiotrophoblast layers of WT, <i>Vash1^(−/−)</i> and <i>Vash2^(−/−)</i> mice.

<p>A: Semi-thin sections of the labyrinth layer of WT, <i>Vash1^(−/−)</i>, and <i>Vash2^(−/−)</i> placentas. Bar = 20 µm. B: Electron microscopic pictures of WT and <i>Vash2^(−/−)</i> placentas. Purple indicates ECs; green, ST-I; and yellow, ST-II. Bar = 5 µm. C–E: Expression of Gcm-1, Syn-B, and Syn-A in WT, <i>Vash1^(−/−)</i> and <i>Vash2^(−/−)</i> placentas at the indicated dpc, was determined by qRT-PCR. At 12.5, 16.5, and 18.5 dpc, the respective placenta numbers were 7, 6, and 5 for WT; 7, 7, and 7 for <i>Vash1^(−/−)</i>; and 5, 7, and 6 for <i>Vash2^(−/−)</i>. *P<0.01, NS; not significant.</p

Relationship between MAP and LSFG parameters.

<p>Correlation between MAP and LSFG measurements were analyzed for MBR (A), BOS (B), FAI (C), and RI (D). Pearson correlation coefficients were used to assess relationships between MAP and each measurement variable. MAP: mean arterial pressure, MBR: mean blur rate, BOS: Blowout score, FAI: Flow acceleration index, RI: Resistive index.</p

Baseline characteristics of participants.

<p>Baseline characteristics of participants.</p

Longitudinal changes of LSFG parameters throughout pregnancy.

<p>Assessment of LSFG measurements at each time point (T1. 11–13 weeks, T2. 19–21 weeks, T3. 28–30 weeks, T4. 34–36 weeks) for MBR (A), BOS (B), FAI (C), and RI (D). Generalised linear mixed model was used to determine level of significance versus T1. * = p<0.05. MBR: mean blur rate, BOS: Blowout score, FAI: Flow acceleration index, RI: Resistive index.</p