Measurement of free and complexed soluble vascular endothelial growth factor receptor, Flt-1, in fluid samples: development and application of two new immunoassays

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.

Vascular endothelial growth factor and tissue factor in patients with established peripheral artery disease: a link between angiogenesis and thrombogenesis?

“…The present study is larger than previous work in PAD [10,11], and we have also extended observations by Roller et al [11] by measuring sFlt-1 levels in this patient population. In our subgroup analyses (Table 5), we did not find any associations with smoking, previous hypertension, ischaemic heart disease, stroke, antiplatelet therapy or hyperlipidaemia in our cohort of PAD patients.…”

“…Furthermore, circulating sFlt-1 may serve as a selective inhibitor of VEGF activity and this regulatory mechanism may be altered by pathological conditions [10]. Indeed, levels of sFlt-1 are also significantly correlated with both VEGF and TF, although the exact significance of this is unclear as the difference in sFlt-1 levels between patients and controls was not statistically significant.…”

“…Nevertheless, the role of the VEGF receptor soluble Flt1 (sFlt1) in PAD and the relation to tissue factor (TF) is unknown. sFlt-1 levels have been reported to be decreased in patients with arterial disease [10], although the significance of this is not clear.…”

“…In ischaemic disease, VEGF is stimulated by hypoxia [7], which is evident in tissue ischaemia. Furthermore, VEGF has been demonstrated to be increased in risk factors for vascular disease, such as hypertension [8], atherosclerosis with hyperlipidaemia [9] and PAD [10,11]. Nevertheless, the role of the VEGF receptor soluble Flt1 (sFlt1) in PAD and the relation to tissue factor (TF) is unknown.…”

Soluble vascular endothelial growth factor, soluble VEGF receptor Flt-1 and endothelial function in healthy smokers

“…This ELISA system has inter-and intra-coefficient of variation less than 10% and 5%, respectively [14]. Levels of VEGF were measured from citrate plasma with a commercially available ELISA (R&D Systems, Minneapolis, MN).…”