Release and complex formation of soluble VEGFR-1 from endothelial cells and biological fluids

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 (PIGF), 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 PIGF 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 VEGF165 and PIGF-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.     

Quantification of vascular endothelial growth factor-C (VEGF-C) by a novel ELISA

Lymphangiogenesis plays an important role in several normal and pathological conditions such as wound healing, inflammation or metastasis formation in several malignancies. VEGF-C and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. In order to develop a highly sensitive and specific detection system for VEGF-C, we produced soluble binding proteins and antibodies for a microtiterplate-based assay. Here we describe a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of human, rat and murine VEGF-C. The different antibodies developed against human and rat VEGF-C could be combined to detect processed and partially processed VEGF-C in a specific way. The ELISA was able to detect human and rat VEGF-C with a minimum detection limit of 100 pg/ml. The assay did not show any cross-reactivity with the related protein VEGF-D. Furthermore, complex formation with its soluble receptors VEGFR-2 and VEGFR-3 did not restricted the sensitivity of the assay. Using this assay, VEGF-C was measured in supernatants and lysates of different cell types and in tumour tissue samples of murine, rat and human origin. Cell lines secrete VEGF-C in very low amounts (<1 ng/ml) whereas VEGF-C transfected cells can secrete up to 50 ng/ml VEGF-C into the supernatant. In human tumour tissue samples VEGF-C was detected in some carcinomas in the low protein range. This ELISA will be a useful tool for investigations concerning the physiological function of VEGF-C in lymphangiogenesis under normal and pathophysiological conditions.     

Antiangiogenic effect of soluble vascular endothelial growth factor receptor-1 in placental angiogenesis.

Differential splicing of the flt-1 mRNA generates soluble variant of vascular endothelial growth factor (VEGF) receptor-1 (sVEGFR-1, also known as sFlt-1). The action of VEGF is antagonized by sVEGFR-1. Soluble VEGFR-1 binds to VEGF with a high affinity and therefore works to modulate VEGF and VEGF signaling pathway. In this study, the authors tested the hypothesis that VEGF-mediated endothelial cell angiogenesis is tightly modulated by the release of sVEGFR-1 and placental expression of sVEGFR-1 is upregulated by hypoxia. Immunolocalization studies showed progressively intense staining for sVEGFR-1 and VEGF in the trophoblast of placental villous explants throughout gestation. Endothelial cell migration studies using a modified Boyden's chamber showed a significant increase in cell migration in response to VEGF that was significantly attenuated in the presence of exogenous sVEGFR-1. Furthermore, stimulation of endothelial cells with VEGF led to a dose-dependent increase in the release of sVEGFR-1 as determined by enzyme-linked immunosorbent assay (ELISA). Exposure of normal placental villous explants to hypoxia (1% pO2) increased trophoblast expression of sVEGFR-1 when compared with tissue normoxia (5% pO2). In addition, conditioned media from hypoxia treated placental villous explants induced a significant increase in endothelial cell migration that was significantly reduced in presence of sVEGFR-1. Our study demonstrates that hypoxia positively regulates sVEGFR-1 protein expression in ex vivo trophoblasts, which control VEGF-driven angiogenesis.     

Poor responder-high responder: the importance of soluble vascular endothelial growth factor receptor 1 in ovarian stimulation protocols

This study was designed to detect vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-1) in follicular fluid specimens and to evaluate the importance of sVEGFR-1 with respect to ovarian response to gonadotrophin stimulation. A total of 69 patients was treated for IVF with recombinant human follicle stimulating hormone (FSH). Concentrations of VEGF and sVEGFR-1 were quantified in follicular fluids from oocyte retrievals. Patients were designated to three groups with respect to the number of harvested oocytes: group A, 1-5 oocytes; group B, 6-10 oocytes; group C, >10 oocytes. In group A, 1133 +/- 870 pg VEGF/ml follicular fluid per oocyte were quantified, in group B 426 +/- 262 pg VEGF/ml per oocyte, and in group C 274 +/- 179 pg VEGF/ml per oocyte. Soluble VEGFR-1 concentrations resulted in 1200 +/- 523 pg/ml follicular fluid per oocyte in group A, 255 +/- 193 pg/ml per oocyte in group B, and 79 +/- 69 pg/ml per oocyte in group C. No free sVEGFR-1 could be detected in any follicular fluid. An index to estimate the biological activity of VEGF by dividing VEGF/sVEGFR-1 revealed an increasing availability of VEGF with higher ovarian response to gonadotrophin therapy. In group A this index was 1.03, in group B 1.71, and in group C 3.21. A delicate balance between VEGF and sVEGFR-1 is necessary to allow an adequate ovarian reaction to gonadotrophin therapy. Excess of bio-active VEGF increases the risk for ovarian hyperstimulation syndrome. Excess of sVEGFR-1 results in poor response and goes in parallel with reduced chances for conception.     

Soluble and Membranous Vascular Endothelial Growth Factor Receptor-2 in Pregnancies Complicated by Pre-Eclampsia

Purpose

There is a paucity of information on the serum soluble vascular endothelial growth factor receptor-2 (sVEGFR-2) concentrations, membranous VEGFR-2 expression and the mechanism involved in their modulations during the clinical onset of pre-eclampsia. This cross-sectional study was conducted to evaluate the concentration of sVEGFR-2 in serum and to investigate the expression of membranous VEGFR-2 in placentae of pre-eclampsia group.

Materials and Methods

The serum levels of sVEGFR-2 (n = 120) and the expression of membranous VEGFR-2 in placentae (n = 100) were analysed at third trimester of pregnancy by enzyme linked immunosorbent assay (ELISA) and immunohistochemistry respectively. The diagnostic parameters of sensitivity, specificity and association of soluble and membranous VEGFR-2 in these patients were evaluated.

Results

The serum levels of sVEGFR-2 in pre-eclampsia patients were found to be significantly reduced (p = 0.01, p = 0.001) in early and late pre-eclamptic sub-groups as compared to their respective third trimester control sub-groups. Also, the receiver operating characteristic (ROC) curve analysis showed a cut-off value of 7350.4 pg/mL, higher sensitivity (76%) and specificity (76%) for sVEGFR-2 in late onset (> 34 weeks) pre-eclamptic group. Significant down-regulation of membranous VEGFR-2 immunoreactivity was observed in all the placental cells (p = 0.0001) at > 34 weeks preeclamptic group.

Conclusion

The reduced serum levels of soluble VEGFR-2 and the down-regulated expression of membranous VEGFR-2 in the study group denoted abnormality in VEGF mediated placental function in all placental cells and thus VEGFR-2 may be a key factor, intimately associated with pre-eclampsia. This study shows the clinical utility of soluble and membranous VEGFR-2 in pre-eclampsia patients.     

Quantification and detection of DcR3, a decoy receptor in TNFR family

A soluble decoy receptor, DcR3, belongs to the tumor necrosis factor receptor (TNFR) family, and this receptor is known to bind to three TNF family ligands, namely Fas ligand (FasL), LIGHT, and TL1A. To aid our understating of the role of DcR3 in the immune system, we have developed quantitative enzyme-linked immunosorbent assay (ELISA) to detect soluble DcR3 in human biological fluids. Two monoclonal antibodies, MD3E2 and MD3B1, that recognized different epitopes on the DcR3 molecule were selected as capture and detection antibodies, respectively, to be paired in ELISA. The assay had a detection limit of 36 pg/ml with a dynamic range of 0.25-16 ng/ml. The recovery range was 91-112% for cell culture supernatant and 90-108% for human sera. Intra- and inter-assay CVs were less than 7.2% and 11.2%, respectively. Among a panel of cell lines tested, colon adenocarcinoma cell line, SW480, secreted the highest levels of DcR3 at 3.2 ng/ml. From the screening of human sera samples, we discovered that 39 healthy individuals, 59 tumor patients, and 46 patients with renal failure expressed an average (mean+/-S.D.) 0.56+/-0.52, 2.3+/-1.6, and 4.6+/-2.8 ng/ml DcR3, respectively. To confirm the specificity of ELISA, we have purified native DcR3 from SW480 cell culture supernatants and identified a native DcR3 in a clinical serum by immunoprecipitation. Taken together, our data demonstrated that the ELISA developed in this study was specific and sensitive to quantify soluble DcR3 in a variety of human biological fluids and that the assay would be useful for studying the regulation of DcR3 in certain pathophysiological conditions.     

Fetal and maternal angiogenic/anti-angiogenic factors in normal and preeclamptic pregnancy

Few studies evaluated angiogenic/anti-angiogenic factors and endothelial (dys)function in both maternal and umbilical cord blood (UCB) in preeclampsia (PE). We aimed to clarify the role of placental growth factor (PlGF), vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor 1 (VEGFR-1) and tissue plasminogen activator (tPA), by evaluating them in maternal and UCB in 42 normal and 46 preeclamptic (PEc) cases.

In PE, maternal and UCB PlGF were significantly lower; maternal VEGF, sVEGFR-1 and tPA were significantly higher. In UCB, sVEGFR-1 and tPA were significantly higher in PEc cases, while VEGF and PlGF were significantly lower. A significant correlation between maternal and UCB sVEGFR-1, and between sVEGFR-1 and tPA both in maternal and UCB, was observed in PEc cases.

In maternal and UCB circulation in PE, a close interaction seems to exist between endothelial dysfunction and angiogenesis disturbance, and sVEGFR-1 seems to play a central role in those disturbances.     

Anti-VEGFR-2 scFvs for cell isolation. Single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/flk-1) on the surface of primary endothelial cells and preselected CD34+ cells from cord blood

Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.     

GM-CSF induces expression of soluble VEGF receptor-1 from human monocytes and inhibits angiogenesis in mice

GM-CSF promotes homeostasis of myeloid cells. We report that GM-CSF upregulates mRNA and protein production of the soluble form of membrane bound VEGF receptor-1 (sVEGFR-1) in human monocytes. This sVEGFR-1 was biologically active, as cell-free supernatants from GM-CSF-stimulated monocytes blocked detection of endogenously expressed VEGF and inhibited endothelial cell migration and tube formation, even in the presence of exogenous rhVEGF. VEGF activity was recovered by neutralizing sVEGFR-1. To determine whether these events were important in vivo, Matrigel plugs were incubated with rhVEGF, rhGM-CSF, or rhGM-CSF/rhVEGF and injected into mice. Plugs containing GM-CSF or GM-CSF/VEGF had less endothelial cell invasion than plugs containing rhVEGF and were similar to plugs incubated with PBS alone. Neutralizing antibodies specific for sVEGFR-1 injected in these plugs reversed the effects of GM-CSF or GM-CSF/VEGF, while an isogenic antibody did not. Thus, GM-CSF and monocytes play a vital role in angiogenesis through the regulation of VEGF and sVEGFR-1.     

An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7-104%). The working range is 0.15-34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269-299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes.     

Vascular Endothelial Growth Factor Receptor-1 Modulates Vascular Endothelial Growth Factor-Mediated Angiogenesis via Nitric Oxide

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.     

Identification of naturally secreted soluble form of TL1A, a TNF-like cytokine

TL1A, a TNF-like ligand, mediates signaling via its cognate receptor DR3, a death receptor whose activation was known to induce both death and survival factors. TL1A, like TNF, is also presumed to circulate as a homotrimeric soluble form. To identify soluble TL1A in the immune system, we have developed a quantitative ELISA by pairing a monoclonal antibody (MAb) with a biotinylated anti-TL1A polyclonal antibody (PAb) as capture and detection antibodies, respectively. The assay had a detection limit of 32 pg/ml. Overnight culture of human umbilical vein endothelial cells (HUVEC) expressed up to 160 pg/ml of TL1A, and that proinflammatory cytokines, IL-1 and TNF, significantly increased TL1A mRNA and soluble protein up to several folds in contrast to IL-6 and IL-11, which induced neither protein nor mRNA in the cells. IL-1 appeared to be a better stimulator of TL1A than TNF-alpha, and the induction of soluble TL1A in HUVEC in response to IL-1 was dose and time-dependent. We have also purified soluble TL1A from a large volume of IL-1 stimulated HUVEC conditioned medium using an anti-TL1A PAb-coupled affinity column. The protein eluted from the column was further reacted with anti-TL1A MAbs in Western blot: 30-kDa and 32-kDa under non-reducing and reducing conditions, respectively. Taken together, our results indicated that ELISA might be useful in studying soluble TL1A regulation in certain inflammatory conditions.     

Transcription and translation of human F11R gene are required for an initial step of atherogenesis induced by inflammatory cytokines

Background

Several proteins that promote angiogenesis are overexpressed in hepatocellular carcinoma (HCC) and have been implicated in disease pathogenesis. Sunitinib has antiangiogenic activity and is an oral multitargeted inhibitor of vascular endothelial growth factor receptors (VEGFRs)-1, -2, and -3, platelet-derived growth factor receptors (PDGFRs)-α and -β, stem-cell factor receptor (KIT), and other tyrosine kinases. In a phase II study of sunitinib in advanced HCC, we evaluated the plasma pharmacodynamics of five proteins related to the mechanism of action of sunitinib and explored potential correlations with clinical outcome.

Methods

Patients with advanced HCC received a starting dose of sunitinib 50 mg/day administered orally for 4 weeks on treatment, followed by 2 weeks off treatment. Plasma samples from 37 patients were obtained at baseline and during treatment and were analyzed for vascular endothelial growth factor (VEGF)-A, VEGF-C, soluble VEGFR-2 (sVEGFR-2), soluble VEGFR-3 (sVEGFR-3), and soluble KIT (sKIT).

Results

At the end of the first sunitinib treatment cycle, plasma VEGF-A levels were significantly increased relative to baseline, while levels of plasma VEGF-C, sVEGFR-2, sVEGFR-3, and sKIT were significantly decreased. Changes from baseline in VEGF-A, sVEGFR-2, and sVEGFR-3, but not VEGF-C or sKIT, were partially or completely reversed during the first 2-week off-treatment period. High levels of VEGF-C at baseline were significantly associated with Response Evaluation Criteria in Solid Tumors (RECIST)-defined disease control, prolonged time to tumor progression (TTP), and prolonged overall survival (OS). Baseline VEGF-C levels were an independent predictor of TTP by multivariate analysis. Changes from baseline in VEGF-A and sKIT at cycle 1 day 14 or cycle 2 day 28, and change in VEGF-C at the end of the first off-treatment period, were significantly associated with both TTP and OS, while change in sVEGFR-2 at cycle 1 day 28 was an independent predictor of OS.

Conclusions

Baseline plasma VEGF-C levels predicted disease control (based on RECIST) and were positively associated with both TTP and OS in this exploratory analysis, suggesting that this VEGF family member may have utility in predicting clinical outcome in patients with HCC who receive sunitinib.

Trial registration

ClinicalTrials.gov: NCT00247676     

1Implication of soluble receptors of VEGF, sVEGFR-1 and sVEGFR-2, in angiogenesis

Introduction:  sVEGFR-1 and sVEGFR-2 are soluble forms of the membrane-bound receptors of VEGF. sVEGFR-1 is detected in plasma of pre-eclamptic women, during ischemia and in some cancer cases. sVEGFR-2, was recently detected in plasma of healthy people, in leukaemia and in systemic erythematosus lupus cases. sVEGFR-1 has anti-angiogenic properties in vitro and in vivo but sVEGFR-2 remains uncharacterized and its physiological or pathological role is still unknown.
Material and Methods:  The aim of this study was to understand and to characterize the role of sVEGFR2 in angiogenesis and in endothelial function.
Results:  In aortic ring assay, an ex vivo model of angiogenesis, sVEGFR1 and sVEGFR2 were able to abolish VEGF-induced angiogenesis. However, when used alone, they induced the formation of a "network", supposed to be vascular in visible microscopy. As they were able to abolish the effect of VEGF on endothelial function but showed no direct effect alone, we performed an immuno-staining of the "vascular network" induced by the soluble receptors. It showed that there were a few endothelial cells but mostly pericytes/smooth muscle cells (PC/SMC). Our first in vitro experiments on PC/SMC showed that sVEGFR-1 and sVEGFR-2 were able to promote the migration of PC/SMC, only in presence of endothelial cells.
Conclusions:  Our results evidence that sVEGFR1 and sVEGFR2 inhibit VEGF-induced angiogenesis in a similar way. However, they have also a direct effect on PC/SMC, promoting their migration. Our results suggest that these soluble receptors could act, not only on endothelial cells themselves, but by a direct effect on PC/SMC too. These results contribute to identify factors by which it could be possible to regulate the balance between pro-angiogenic and anti-angiogenic factors, especially in the case of the anti-VEGF drugs used now as anti-cancer therapies in clinics, where a transient "normalization" of the vessels is observed.     

A novel monoclonal antibody screening method using the Luminex-100 microsphere system

We describe the development of a robust and sensitive assay system (detection limit <500 pg/ml biotin-IL-6, K(d)=75 ng/ml), using Luminex-100 microspheres, that could effectively screen for neutralizing antibody whenever a soluble form of the receptor for a target molecule is available. As an example, we coupled a recombinant human interleukin-6 soluble receptor to a Luminex carboxylated microsphere and used a biotin-labeled recombinant human interleukin-6 as a probe to assess binding competition. Three anti-human IL-6 monoclonal antibodies that bind distinct IL-6 epitopes were used as test articles to evince the stringency of the screen. Our assay was able to detect antibody concentration as low as 10 ng/ml without interference from hybridoma growth medium or cell supernatant. The time-saving benefits of this assay format make it ideal for high-throughput screening (HTS) applications for neutralizing monoclonal antibodies.     

No soluble common cytokine receptor gamma chain (gamma(c)) in activated human lymphocyte cultures-comparison with soluble IL-2Ralpha

The presence of a soluble form of the common cytokine receptor gamma chain (gamma(c)) in cell free supernatants from unstimulated and 1-6 days PHA stimulated peripheral blood lymphocyte (PBL) cultures was analyzed using a sandwich ELISA. No naturally produced soluble gamma(c) could be detected in these cell free culture supernatants, although a sensitivity in the nanogram range was achieved for recombinant baculovirus expressed human soluble gamma(c) with this assay (detection limit 0.2 ng hIL-2 sRgamma). Analysis of the very same supernatants for soluble IL-2Ralpha demonstrated increased concentrations (up to 20.4 ng/ml) of this other IL-2R member. The membrane-associated form of the common cytokine receptor gamma chain was detected in cell lysates prepared from stimulated PBL at a concentration of 3.5 ng per 0.5 x 10(6) cells. Analysis of a small panel of serum samples from patients with different disorders verified that the soluble form of hIL-2Ralpha, but not hIL-2 sRgamma, can be detected, which thereby strongly suggests that the human soluble gamma(c) seems to be a valuable marker only for a limited number of clinical disorders.     

Circulating protein biomarkers of pharmacodynamic activity of sunitinib in patients with metastatic renal cell carcinoma: modulation of VEGF and VEGF-related proteins

Background

Sunitinib malate (SUTENT^®) is an oral, multitargeted tyrosine kinase inhibitor, approved multinationally for the treatment of advanced RCC and of imatinib-resistant or – intolerant GIST. The purpose of this study was to explore potential biomarkers of sunitinib pharmacological activity via serial assessment of plasma levels of four soluble proteins from patients in a phase II study of advanced RCC: VEGF, soluble VEGFR-2 (sVEGFR-2), placenta growth factor (PlGF), and a novel soluble variant of VEGFR-3 (sVEGFR-3).

Methods

Sunitinib was administered at 50 mg/day on a 4/2 schedule (4 weeks on treatment, 2 weeks off treatment) to 63 patients with metastatic RCC after failure of first-line cytokine therapy. Predose plasma samples were collected on days 1 and 28 of each cycle and analyzed via ELISA.

Results

At the end of cycle 1, VEGF and PlGF levels increased >3-fold (relative to baseline) in 24/54 (44%) and 22/55 (40%) cases, respectively (P < 0.001). sVEGFR-2 levels decreased ≥ 30% in 50/55 (91%) cases and ≥ 20% in all cases (P < 0.001) during cycle 1, while sVEGFR-3 levels were decreased ≥ 30% in 48 of 55 cases (87%), and ≥ 20% in all but 2 cases. These levels tended to return to near-baseline after 2 weeks off treatment, indicating that these effects were dependent on drug exposure. Overall, significantly larger changes in VEGF, sVEGFR-2, and sVEGFR-3 levels were observed in patients exhibiting objective tumor response compared with those exhibiting stable disease or disease progression (P < 0.05 for each analyte; analysis not done for PlGF).

Conclusion

Sunitinib treatment in advanced RCC patients leads to modulation of plasma levels of circulating proteins involved in VEGF signaling, including soluble forms of two VEGF receptors. This panel of proteins may be of value as biomarkers of the pharmacological and clinical activity of sunitinib in RCC, and of angiogenic processes in cancer and other diseases.     

Establishment of an ELISA system for determining soluble LAIR-1 levels in sera of patients with HFRS and kidney transplant

LAIR-1, the leukocyte-associated Ig-like receptor-1, is a trans-membrane molecule that functions as an inhibitory receptor on natural killer cells, T lymphocytes and monocytes. It has been well known that many trans-membrane receptors can shed from the cell surface and be released into the circulation in soluble form when lymphocytes, endothelials and other immune cells are activated. In many cases, the levels of soluble receptors in the circulation can be used as markers of lymphocyte activation in transplant patients and virus infection patients. To investigate whether LAIR-1 is able to be released into the sera, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) system based on two anti-LAIR-1 monoclonal antibodies (MAb) with different epitope specificities. Using this ELISA, we found that sLAIR-1 existed in the supernatants collected from PMA, PHA or CD3 MAb-stimulated lymphocytes cultures in vitro for the first time. Moreover, we found that LAIR-1 level in serum samples from healthy individuals was 6.2 +/- 3.3 ng/ml, whereas the levels in sera of patients with hemorrhagic fever with renal syndrome (HFRS) and patients 3-7 days after kidney transplant increased to 47.2 +/- 35.9 and 24.4 +/- 16.0 ng/ml, respectively. Furthermore, HFRS patients in oliguric phase showed higher serum sLAIR-1 levels than those in other phases, and transplant patients with rejection showed higher serum sLAIR-1 level than those without rejection. These findings demonstrated that LAIR-1 can be released when lymphocytes are activated, suggesting sLAIR-1 may be used as a predictor for monitoring immune reaction in some virus infections and organ transplants which may be useful in clinical treatment of these diseases.     

Soluble forms of membrane cofactor protein (CD46, MCP) are present in plasma, tears, and seminal fluid in normal subjects.

We have established an ELISA for determination of membrane cofactor protein (MCP, CD46) both solubilized from cell membranes and released in body fluids. In this assay, mouse MoAbs against MCP, M177 and M160 whose epitopes were different, were used as capture and detection antibodies, respectively. The NP-40 concentration in samples for MCP to be measured must be less than 0.05%. The detection limit of this MCP assay was 0.5 ng. The assay was used to quantify solubilized membrane MCP, and soluble MCP in normal human plasma, serum, urine, saliva, tears, and seminal fluid, and culture media of tumour cell lines. Soluble MCP was barely detected in the conditioned media of the cell lines. The levels of sMCP in plasma and serum were 10-60 ng/ml and that in tears, 0-50 ng/ml. Seminal fluid contained about 10-fold more soluble MCP than serum. Soluble MCP was not detectable by this assay in the other body fluids, suggesting that their MCP levels were less than the detection limit, if any.     

Construction of ELISA system to quantify human ST2 protein in sera of patients

The human ST2 gene can be specifically induced by growth stimulation in fibroblastic cells, and can also be induced by antigen stimulation in Th2 cells. The gene encodes a soluble secreted protein, ST2, and a transmembrane protein, ST2L, which are closely related to the interleukin-1 receptor. To gain insight into the biological roles of the ST2 gene, three monoclonal antibodies (MAbs) against human ST2 gene products were obtained. To obtain these antibodies, immunization was carried out using two different immunogens: purified soluble human ST2 protein (hST2), and COS7 cells, which express the extracellular portion of human ST2L. 2A5 and FB9 MAbs were derived from the immunization with soluble hST2, and HB12 was derived from the COS7 cell immunization. All three antibodies were shown to detect native forms of the human ST2 gene products by immunoprecipitation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In the competitive ELISA using biotinylated and nonlabelled MAbs, neither FB9 nor HB12 affected the binding of 2A5 to ST2 gene products. Based on this result, we constructed a sandwich ELISA system using 2A5 and FB9 to measure the concentration of soluble hST2 in sera. The ELISA, combined with the flow cytometry using these antibodies, will be a useful tool for elucidating the functions of human ST2 gene products in individuals.