Release and complex formation of soluble VEGFR-1 from endothelial cells and biological fluids

One of the key molecules promoting angiogenesis is the endothelial cell-spe cific mitogen, vascular endothelial growth factor (VEGF or VEGF-A), which a cts through two high-affinity receptor tyrosine kinases (VEGFR), VEGFR-1 (o r Flt-1) and VEGFR-2 (or KDR/Flk-1). It was shown before that a soluble var iant of VEGFR-1 (sVEGFR-1) can be generated by differential splicing of the fit-1 mRNA. This soluble receptor is an antagonist to VEGF action, reducin g 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 gro wth retardation. Here we show that sVEGFR-1 is produced by cultured human m icrovascular and macrovascular endothelial cells and a human melanoma cell line. The soluble receptor is mainly complexed with ligands; only 5-10% rem ains detectable as free, uncomplexed receptor protein. Furthermore, we show the time course of total and free sVEGFR-1 release together with its putat ive ligands, VEGF-A and placenta growth factor (PIGF), from macrovascular e ndothelial cells. The release of sVEGFR-1 was quantitatively measured in tw o different ELISA types. The release of sVEGFR-1 was strongly enhanced by p horbol-ester (PMA); the cells produced up to 22 ng/ml of sVEGFR-1 after 48 hours. The expression of VEGF-A and PIGF was moderately influenced by PMA. We also show a hypoxia-induced increase of sVEGFR-1 expression in cells cul tured from placenta, a tissue that has a high flt-1 gene expression. Moreov er, we demonstrate that sVEGFR-1 in amniotic fluids acts as a sink for exog enous VEGF(165) and PIGF-2. Here, for the first time, to what extent recomb inant ligands have to be added to compensate for the sink function of amnio tic fluids was analyzed. In conclusion, human endothelial cells produce hig h levels of sVEGFR-1, which influences the availability of VEGF-A or relate d ligands. Therefore, sVEGFR-1 may reduce the ligand binding to transmembra ne receptors and interfere with their signal transduction.