Detection and quantification of complexed and free soluble human vascular endothelial growth factor receptor-1 (sVEGFR-1) by ELISA

Hornig, Carsten; Behn, Tanja; Bartsch, W.; Yayon, Avner; Weich, Herbert A.

doi:10.1016/s0022-1759(99)00065-4

Cited by 49 publications

(40 citation statements)

References 16 publications

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“…Based on these results, we collected serum-free CM of different cell types known to produce sVEGFR-1. By ELISA measurements, we were able to confirm that CM from primary microvascular and macrovascular endothelial cell and melanoma cell line contains sVEGFR-1 in different amounts (Hornig et al, 1999). With the Far-Western and cross- Effect of oxygen concentration gradient on sVEGFR-1 levels in CM of cultured placenta villi.…”

Section: Discussionmentioning

confidence: 71%

“…The CM were examined for their sVEGFR-1 content in two different ELISA types, designed to recognize either the total amount of sVEGFR-1 (Hornig et al, 1999), or free, uncomplexed sVEGFR-1 only. The results are shown in Fig.…”

Section: Kinetics Of Svegfr-1 Vegf-a and Plgf Release From Culturedmentioning

confidence: 99%

“…We designed experiments with amniotic fluids, which contain large amounts of endogenous sVEGFR-1 (Hornig et al, 1999), to obtain more information on the possible activity and complex status of sVEGFR-1 harvested from a biological sample. Pooled amniotic fluids (14 th to 16 th week of gestation) were first measured in both sVEGFR-1 ELISA types.…”

Section: Svegfr-1 In Amniotic Fluids Functions As a Sink For Vegf-a Amentioning

confidence: 99%

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

Hornig¹,

Barleon

Ahmad

et al. 2000

Laboratory Investigation

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134

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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).

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Section: Discussionmentioning

confidence: 71%

Section: Kinetics Of Svegfr-1 Vegf-a and Plgf Release From Culturedmentioning

confidence: 99%

Section: Svegfr-1 In Amniotic Fluids Functions As a Sink For Vegf-a Amentioning

confidence: 99%

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

Hornig¹,

Barleon

Ahmad

et al. 2000

Laboratory Investigation

Self Cite

205

134

View full text Add to dashboard Cite

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“…The minimal detection limit for total VEGF was 31 pg ml À1 . A receptor -ligand detection assay was applied to detect free bioactive VEGF following the basic protocol for total VEGF ELISA, except that plates were coated with 0.5 mg ml À1 sVEGFR-1 (D1 -D6) produced in insect cells (Hornig et al, 1999). This ensured that no VEGFR-1 complex forms were recognised.…”

Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

Association between intratumoral free and total VEGF, soluble VEGFR-1, VEGFR-2 and prognosis in breast cancer

et al. 2005

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Vascular endothelial growth factor (VEGF) receptors consist of three cell-membrane type receptors (VEGFR-1, VEGFR-2 and VEGFR-3), and soluble form of VEGFR-1 (sVEGFR-1), an intrinsic negative counterpart of the VEGF. In this study, we measured intratumoral protein levels of free and total VEGF, VEGFR-2 and sVEGFR-1 from 202 primary breast cancer tissues and examined their prognostic values. A significant inverse correlation was found between free or total VEGF and oestrogen receptor (ER) status ( P =0.042 and 0.032, respectively). A univariate analysis showed that low sVEGFR-1 and high total VEGF were significantly associated with poor prognosis in disease-free survival (DFS) and overall survival (OS). The ratio of sVEGFR-1 to total VEGF was a strong prognostic indicator (DFS: P =0.008; OS: P =0.0002). A multivariate analysis confirmed the independent prognostic values of total VEGF and the ratio of sVEGFR-1 to total VEGF. In subgroup analysis, total VEGF was a significant prognostic indicator for ER-positive tumours but not for ER-negative tumours, whereas sVEGFR-1 was significant for ER-negative tumours but not for ER-positive tumours. In conclusion, the intratumoral sVEGFR-1 level, VEGF level and the ratio of sVEGFR-1 to total VEGF are potent prognostic indicators of primary breast cancer, and might be relevant to ER status.

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“…14 The VEGFR-1 sandwich immunoassay used 1 monoclonal anti-human VEGFR-1 antibody and 1 polyclonal antibody. 22,23 …”

Section: Measurement Of Total-vegf-a Free-vegf-a Vegf-c and Vegfr-1mentioning

confidence: 99%

Immunodetection and quantification of vascular endothelial growth factor receptor‐3 in human malignant tumor tissues

Bando

Brokelmann

Toi

et al. 2004

Intl Journal of Cancer

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Vascular endothelial growth factor recpetor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme-linked immunosorbent assay (ELISA) for the quantification of VEGFR-3 in different human cell and tissue lysates. A combination of the goat polyclonal anti-VEGFR-3 antibody and the mouse monoclonal anti-human VEGFR-3 antibody was used. The assay was highly sensitive and reproducible with a detection range of 0.2-25 ng/ ml. The assay was specific for VEGFR-3, with no crossreactivity to VEGFR-1 or VEGFR-2. Complex formation with VEGF-C and VEGF-D had no effect on the sensitivity of the assay. The VEGFR-3 concentration in the lysates of cultured human dermal microvascular endothelial cells was 14-fold higher than in the lysates from human umbilical vein endothelial cells. In human kidney, breast, colon, gastric and lung cancer tissues the protein levels of VEGFR-3 were in the range of 0.6 -16.7 ng/mg protein. Importantly, the level of VEGFR-3 protein detected in the ELISA correlated significantly with the number of VEGFR-3 positive vessels observed in histochemical sections, suggesting that the ELISA assay may be a reliable surrogate of measuring VEGFR-3-positive vessel density. The protein levels of VEGFR-3 in 27 renal cell carcinoma samples had a significant correlation with the levels of VEGF-C (p<0.001), or biological active, free VEGF-A (p<0.0001), but not with VEGFR-1 or total VEGF-A. This assay provides a useful tool for the investigations of the expression levels of VEGFR-3 in physiological and pathological processes, particular in cancer and in lymphangiogenesisrelated disease.

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Detection and quantification of complexed and free soluble human vascular endothelial growth factor receptor-1 (sVEGFR-1) by ELISA

Cited by 49 publications

References 16 publications

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

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

Association between intratumoral free and total VEGF, soluble VEGFR-1, VEGFR-2 and prognosis in breast cancer

Immunodetection and quantification of vascular endothelial growth factor receptor‐3 in human malignant tumor tissues

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