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  • Title: Measurement of free and complexed soluble vascular endothelial growth factor receptor, Flt-1, in fluid samples: development and application of two new immunoassays.
    Author: Belgore FM, Blann AD, Lip GY.
    Journal: Clin Sci (Lond); 2001 May; 100(5):567-75. PubMed ID: 11294698.
    Abstract:
    Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. VEGF interacts with the endothelium 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 unknown, with limited data on plasma sFlt-1 levels in disease states. We have developed two new ELISAs for detecting free and VEGF-complexed sFlt-1, which were tested in accordance with standard validation and assessment methodologies employed in commercial settings. The intra-and inter-assay coefficients of variation are <5% and 10% respectively, and results are highly reproducible. Applying these ELISAs in a clinical setting, we measured levels of VEGF, free and complexed sFlt-1 in citrated plasma from 40 patients with cardiovascular 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< or =0.05]. Free sFlt-1 was significantly lower in patients compared with controls [8 ng/ml (2--22 ng/ml) versus 21 ng/ml (10--73 ng/ml), P< or =0.05]. There was no significant difference in the levels of complexed sFlt-1 between the two groups. Plasma levels of VEGF-complexed sFlt-1 are minimal, despite the presence of excess free sFlt-1. Thus unbound plasma VEGF detected by ELISA may represent the majority of circulating VEGF, and justifies the measurement of plasma VEGF as an indicator of circulating VEGF levels. Furthermore, these results suggest that circulating sFlt-1 may serve as a selective inhibitor of VEGF activity, and that this regulatory mechanism may be altered by pathological conditions.
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