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. 2019 Mar 21;10:165. doi: 10.3389/fendo.2019.00165

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Copyright © 2019 Vogtmann, Kühnel, Dicke, Verkaik-Schakel, Plösch, Schorle, Stojanovska, Herse, Köninger, Kimmig, Winterhager and Gellhaus.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Gene expression analysis of important placental labyrinthine markers and Vegf signaling molecules in the hsFLT1/rtTA mouse model. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of marker genes in placentas of each group: fetal growth restriction homozygous (FGR hom; n = 7), FGR heterozygous (het; n = 15), control (n = 12), and doxycycline (Dox) control (n = 13). (A) The mRNA level of fetal endothelial cell marker Cd31 was lower in placentas expressing human soluble fms-like tyrosine kinase 1 (hsFLT1) (FGR hom; FGR het) than in control placentas (control and Dox control). In addition, the expression of syncytiotrophoblast markers, such as gap junction protein connexin 26 (Cx26) and differentiation-promoting transcription factor glial cell missing one (Gcm1), is lower in hsFLT1–expressing placentas (FGR hom more pronounced than FGR het) than in control placentas (control and Dox control). The expression of maternal sinusoidal trophoblast giant cell markers, such as placental alkaline phosphatase (PLAP), was also lower in hsFLT1–expressing placentas than in control placentas, whereas cathepsin Q (Ctsq) expression seemed to be unaffected by hsFLT1 expression in all groups. (B) Murine mRNA levels of sFlt-1 and Flt-1 (m(s)Flt-1) exhibited no clear up- or downregulation between groups (no discrimination between murine sFlt-1 and murine Flt-1 possible at the mRNA level), whereas the expression of murine fetal liver kinase 1 (Flk-1) was lower in FGR hom and FGR het placentas than in control placentas (control and Dox control). Growth factors, such as placental growth factor (Plgf; mainly binding to sFlt-1 and Flt-1), vascular endothelial growth factor A (Vefga; mainly binding to sFlt-1, Flt-1, and Flk-1), and Vegfb (mainly binding to sFlt-1 and Flt-1), are upregulated at the time of hsFLT1 expression (FGR hom and FGR het), a finding correlating with increasing levels of hsFLT1. Nevertheless, the proapoptotic markers caspase9 (Casp9) and Bcl-2–associated death promoter (Bad) downstream of Flk-1 are more highly upregulated upon hsFLT1 expression in FGR hom and FGR het placentas than in either control group (control and Dox control). Samples were obtained from complete placentas at day 18.5 post coitum (dpc). Measured mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh; except for Casp9 and Bad, which were normalized to Flk-1), and control group levels were set at 100% (dotted line). Data is presented as mean ± standard error of the mean. *p < 0.05, **p < 0.01, and ***p < 0.001 determined by the Kruskal–Wallis test with Dunn's post hoc test. (C) Protein levels of Plgf, total Vegf, and Vegfb were reduced upon hsFLT1 expression in FGR hom (n = 5) and het (n = 5) placentas compared to control (n = 5) and Dox control group (n = 5). Data is presented as mean ± standard deviation.