Role of the vasohibin family in the regulation of fetoplacental vascularization and syncytiotrophoblast formation.

Vasohibin-1 (VASH1) and vasohibin-2 (VASH2), the 2 members of the vasohibin family, have been identified as novel regulators of angiogenesis. VASH1 ceases angiogenesis, whereas VASH2 stimulates sprouting. Here we characterized their functional role in the placenta. Immunohistochemical analysis of human placental tissue clarified their distinctive localization; VASH1 in endothelial cells and VASH2 in trophoblasts. We then used a mouse model to explore their function. Wild-type, Vash1((-/-)), and Vash2((-/-)) mice on a C57BL6 background were used in their first pregnancy. As expected, the fetal vascular area was increased in the Vash1((-/-)) mice, whereas it was decreased in the Vash2((-/-)) mice relative to wild-type. In addition, we noticed that the Vash2((-/-)) mice at 18.5dpc displayed thinner villi of the labyrinth and larger maternal lacunae. Careful observation by an electron microscopy revealed that the syncytiotrophoblast formation was defective in the Vash2((-/-)) mice. To test the possible involvement of VASH2 in the syncytiotrophoblast formation, we examined the fusion of BeWo cells, a human trophoblastoid choriocarcinoma cell line. The forskolin treatment induced the fusion of BeWo cells, and the knockdown of VASH2 expression significantly inhibited this cell fusion. Conversely, the overexpression of VASH2 by the infection with adenovirus vector encoding human VASH2 gene significantly increased the fusion of BeWo cells. Glial cell missing-1 and endogenous retrovirus envelope glycoprotein Syncytin 1 and Syncytin 2 are known to be involved in the fusion of trophoblasts. However, VASH2 did not alter their expression in BeWo cells. These results indicate that VASH1 and VASH2 showed distinctive localization and opposing function on the fetoplacental vascularization. Moreover, our study shows for the first time that VASH2 expressed in trophoblasts is involved in the regulation of cell fusion for syncytiotrophoblast formation

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

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

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

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

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

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

Predictive factors for hyperglycaemic progression in patients with schizophrenia or bipolar disorder

Background: Patients with schizophrenia or bipolar disorder have a high risk of developing type 2 diabetes. Aims: To identify predictive factors for hyperglycaemic progression in individuals with schizophrenia or bipolar disorder and to determine whether hyperglycaemic progression rates differ among antipsychotics in regular clinical practice. Method: We recruited 1166 patients who initially had normal or prediabetic glucose levels for a nationwide, multisite, l-year prospective cohort study to determine predictive factors for hyperglycaemic progression. We also examined whether hyperglycaemic progression varied among patients receiving monotherapy with the six most frequently used antipsychotics. Results: High baseline serum triglycerides and coexisting hypertension significantly predicted hyperglycaemic progression. The six most frequently used antipsychotics did not significantly differ in their associated hyperglycaemic progression rates over the 1-year observation period. Conclusions: Clinicians should carefully evaluate baseline serum triglycerides and coexisting hypertension and perform strict longitudinal monitoring irrespective of the antipsychotic used