Release and Complex Formation of Soluble VEGFR-1 from Endothelial Cells and Biological Fluids

Abstract

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).

Changes in Expression of Vascular Endothelial Growth Factor and Its Receptors in Neonatal Hypoxia-Induced Pulmonary Hypertension

“…Similar complexes of 220 kD have been reported in human umbilical vein endothelial cells (12). Furthermore, Hornig et al (31) reported that only 5-10% of the soluble receptor is detected as uncomplexed receptor protein. In pulmonary artery cytosolic fraction, three bands were identified for VEGFR1.…”

Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia

“…free VEGF and free PlGF (22,23). We first confirmed that these ELISA kits measured only unbound VEGF or PlGF by performing a standard curve for VEGF and PlGF proteins in the presence of exogenous recombinant sFlt1.…”

Magnesium sulfate therapy of preeclampsia: an old tool with new mechanism of action and prospect in management and prophylaxis

“…23 Moreover, increased sFlt-1 is suggested to bind to these angiogenic factors and decrease their free fractions. 18,24 Additionally, sFlt-1 competes with VEGFR-2 for VEGF, leading to disturbed angiogenesis, endothelial dysfunction, glomerular damage, impaired glomerular repair and increased apoptosis. 17,25 These changes contribute to the development of hypertension and proteinuria in PE.…”