Measurement of free and complexed soluble vascular endothelial growth factor receptor, Flt-I, in fluid samples: development and application of two new immunoassays

Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogen esis and enhances vascular permeability. VEGF interacts with the endotheliu m via two membrane-spanning receptors, fms-like tyrosine kinase (Flt)-1 and kinase domain receptor. A soluble form of Flt-1 (sFlt-1) was isolated from endothelial cell media; however, its biological significance is still unkn own, with limited data on plasma sFlt-1 levels in disease states. We have d eveloped two new ELISAs for detecting free and VEGF-complexed sFlt-1, which were tested in accordance with standard validation and assessment methodol ogies employed in commercial settings. The intra-and inter-assay coefficien ts of variation are < 5% and 10% respectively, and results are highly repro ducible. Applying these ELISAs in a clinical setting, we measured levels of VEGF, free and complexed sFlt-1 in citrated plasma from 40 patients with c ardiovascular disease and 40 healthy controls. Median (interquartile range) plasma levels of VEGF in patients were significantly greater than controls [403 pg/ml (158-925 pg/ml) versus 113 pg/ml (33-231 pg/ml), P less than or equal to 0.05]. Free sFlt-1 was significantly lower in patients compared w ith controls [8 ng/ml (2-22 ng/ml) versus 21 ng/ml (10-73 ng/ml), P less th an or equal to 0.05]. There was no significant difference in the levels of complexed sFlt-1 between the two groups. Plasma levels of VEGF-complexed sF lt-1 are minimal, despite the presence of excess free sFlt-1. Thus unbound plasma VEGF detected by ELISA may represent the majority of circulating VEG F, and justifies the measurement of plasma VEGF as an indicator of circulat ing VEGF levels. Furthermore, these results suggest that circulating sFlt-1 may serve as a selective inhibitor of VEGF activity, and that this regulat ory mechanism may be altered by pathological conditions.