SUMMARY:One of the key molecules promoting angiogenesis is the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF or VEGF-A), which acts through two high-affinity receptor tyrosine kinases (VEGFR), VEGFR-1 (or Flt-1) and VEGFR-2 (or KDR/Flk-1). It was shown before that a soluble variant of VEGFR-1 (sVEGFR-1) can be generated by differential splicing of the flt-1 mRNA. This soluble receptor is an antagonist to VEGF action, reducing the level of free, active VEGF-A, and therefore, plays a pivotal role in the generation of vascular diseases like pre-eclampsia or intra-uterine growth retardation. Here we show that sVEGFR-1 is produced by cultured human microvascular and macrovascular endothelial cells and a human melanoma cell line. The soluble receptor is mainly complexed with ligands; only 5-10% remains detectable as free, uncomplexed receptor protein. Furthermore, we show the time course of total and free sVEGFR-1 release together with its putative ligands, VEGF-A and placenta growth factor (PlGF), from macrovascular endothelial cells. The release of sVEGFR-1 was quantitatively measured in two different ELISA types. The release of sVEGFR-1 was strongly enhanced by phorbol-ester (PMA); the cells produced up to 22 ng/ml of sVEGFR-1 after 48 hours. The expression of VEGF-A and PlGF was moderately influenced by PMA. We also show a hypoxia-induced increase of sVEGFR-1 expression in cells cultured from placenta, a tissue that has a high flt-1 gene expression. Moreover, we demonstrate that sVEGFR-1 in amniotic fluids acts as a sink for exogenous VEGF 165 and PlGF-2. Here, for the first time, to what extent recombinant ligands have to be added to compensate for the sink function of amniotic fluids was analyzed. In conclusion, human endothelial cells produce high levels of sVEGFR-1, which influences the availability of VEGF-A or related ligands. Therefore, sVEGFR-1 may reduce the ligand binding to transmembrane receptors and interfere with their signal transduction. (Lab Invest 2000, 80:443-454).

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.

Extensive angiogenesis and invasion of the maternal decidua by trophoblasts are essential for the development and function of the placenta. Vascular endothelial growth factors (VEGF), placenta growth factor (PlGF) and their receptors VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt4 have important roles in vasculogenesis and angiogenesis. We have studied the localization of these proteins by immunohistochemistry and Western blotting in the placenta and of PlGF in maternal serum, and their association with diabetes, pre-eclampsia, fetal growth restriction (FGR) and fetal alcohol syndrome (FAS). VEGFR-1 and VEGFR-3 were detected mainly in the syncytiotrophoblastic layer whereas VEGFR-2 was detected in the vascular endothelial cells of the placenta. VEGFR-1, but not the other receptors, showed increased expression in placental syncytiotrophoblasts from 50% of patients with severe pre-eclampsia and FGR when compared with normal placentas. PlGF was undetectable in 38 of 44 samples of amniotic fluid of mothers with normal and complicated pregnancies. However, maternal serum PlGF concentrations were significantly lower in pre-eclamptic patients and in those with FGR when compared to diabetic women or healthy controls. These results suggest that low maternal serum PlGF and increased placental expression of its receptor VEGFR-1 are associated with pre-eclampsia and FGR.

The aetiology of recurrent miscarriage (at least three consecutive miscarriages) usually remains unsolved. The vascular endothelial growth factor (VEGF) family of proteins, together with their receptors and the Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) receptors, are crucial for embryonic development. Therefore, we used immunohistochemistry to analyse the expression of VEGF, the VEGF receptors (VEGFR)-1, -2, and -3, and the Tie-1 and Tie-2 receptors in placental and decidual tissue of women with a history of recurrent miscarriage and missed abortion (MA; n = 12) or blighted ovum (BO; n = 6), and from normal early terminated pregnancies (n = 12). Compared with controls, the MA and BO groups showed: (i) diminished placental trophoblastic VEGF immunoreactivity; (ii) weaker VEGFR-1 and -2 immunoreactivity in decidual vascular endothelium; (iii) reduced placental trophoblastic Tie-1 receptor immunoreactivity; and (iv) reduced decidual vascular endothelial Tie-1 and -2 receptor immunoreactivity. The absence of VEGFR-3 immunoreactivity in decidual vascular endothelium was also noted in all study groups. Interestingly, placental villi from the BO group presented blood vessel-like structures negative for von Willebrand factor, but positive for VEGF, VEGFR-1, -2, -3, Tie-1 and Tie-2 receptor. We conclude that the expression of these antigens may be altered in recurrent miscarriages.

Elevated sVEGFR-1 concentrations at 16-20 wk gestation precede the clinical manifestations of preeclampsia. By neutralizing VEGF, sVEGFR-1 may contribute to inadequate placental vascularization.