Figure 1.
An inverse correlation between VEGFR-2 and sFlt-1 levels in preeclamptic placentae.
A: Representative VEGFR-2 and sFlt-1 immunoblots of PE and PTC placentae. Densitometry analysis comparing normalized VEGFR-2 immunoreactivity between PE (n = 13) and PTC (n = 8) samples. VEGFR-2 transcript levels were assessed by qPCR analysis comparing PE (n = 12) and PTC (n = 7) samples. B: Representative VEGFR-2 and sFlt-1 immunoblots of placental tissues from PE and control (Con) dichorionic twins. (PE, preeclampsia; PTC, age-matched preterm control; *P<0.05). All values are represented as the means±SEM.
Figure 2.
Decreased levels of VEGFR-2 in hypoxia and in early first trimester placentae.
A: Representative VEGFR-2 and sFlt-1 immunoblots of placental villous explants cultured at 3% and 20% O2. Densitometry analysis comparing normalized VEGFR-2 immunoreactivity between explants cultured at 3% (n = 7) and 20% O2 (n = 7). VEGFR-2 transcript levels were assessed by qPCR analysis comparing 3% (n = 4) and 20% O2 (n = 4) samples. B: Representative VEGFR-2 immunoblots of early (EFT) and late (LFT) first trimester placentae. Densitometry analysis comparing normalized VEGFR-2 immunoreactivity between samples were not significantly different. Transcript levels were assessed by qPCR analysis comparing VEGFR-2 levels in EFT and LFT placentae. (EFT, n = 12; LFT, n = 14; *P<0.05). All values are represented as the means±SEM.
Figure 3.
Immunofluorescence localization of sFlt-1 (red) and VEGFR-2 (green).
Nuclear localization by DAPI staining is shown in blue. A: 7 weeks placenta B: 12 week placenta. C–D: control. E–F: early preeclampsia. G: first trimester villous explants at 20% oxygen and H: villous explants at 3% oxygen. Co-immunofluorescence is shown in yellow/orange. White arrow: vessel wall.
Figure 4.
Increased association of VEGFR-2 and sFlt-1 in preeclamptic placentae.
A: Representative sFlt-1 immunoblot of developmental placental samples (two different tissue samples at each gestational age of 8, 12 and 35 weeks) immunoprecipitated with a monoclonal VEGFR-2 antibody. A control lane of protein lysates prepared from placental tissue (35 weeks of gestation) is indicated (Input). B: Representative sFlt-1 immunoblot of PE and PTC samples immunoprecipitated with a monoclonal VEGFR-2 antibody. A negative control lane of PE protein lysate immunoprecipitated in the absence of VEGFR-2 antibody is indicated (NB). Densitometry analysis comparing sFlt-1 immunoreactivity between PE (n = 3) and PTC (n = 6) samples immunoprecipitated with VEGFR-2 antibody. (IP, immunoprecipitation antibody; WB, western blot antibody; PE, preeclampsia; PTC, age-matched preterm control; *P<0.05). All values are represented as the means±SEM.
Figure 5.
sFlt-1 directly attenuates VEGFR-2 expression and downstream signalling in placental villous explants.
A: Representative VEGFR-2, sFlt-1, pAkt and pERK immunoblots of first trimester placental villous explants treated with sFlt-1 or a blocking antibody to sFlt-1. Densitometry analyses comparing normalized VEGFR-2, sFlt-1, pAkt and pERK in treated samples (Con, control; sFlt-1, sFlt-1 protein; α-Flt, Flt-1 neutralizing antibody; n = 3, *P<0.05). All values are represented as the means±SEM. B: Representative VEGFR-2 immunoblot of first trimester placental villous explants treated with VEGF. Densitometry analyses comparing normalized VEGFR-2 between control and VEGF-treated samples (Con, control; VEGF, VEGF-treated; n = 3, *P<0.05). All values are represented as the means±SEM.