Clinical Chemistry 47: 617-623, 2001;
(Clinical Chemistry. 2001;47:617-623.)
© 2001 American Association for Clinical Chemistry, Inc.
Pitfalls in the Measurement of Circulating Vascular Endothelial Growth Factor
Wolfgang Jelkmann1
1
Institut für Physiologie, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. Fax 49-451-500-4151; e-mail
jelkmann{at}physio.mu-luebeck.de.
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Abstract
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Background: Vascular endothelial growth factor (VEGF) is a protein
with antiapoptotic, mitogenic, and permeability-increasing activities
specific for vascular endothelium. VEGF mRNA, which has five isoforms,
is produced by nonmalignant cells in response to hypoxia and
inflammation and by tumor cells in constitutively high concentrations.
Because VEGF plays a crucial role in physiological and
pathophysiological angiogenesis, measurements of circulating VEGF are
of diagnostic and prognostic value, e.g., in cardiovascular failures,
inflammatory diseases, and malignancies. However, there are major
quantitative differences in the published results. This review attempts
to identify reasons for these disparities.
Approach: The literature was reviewed through a Medline search
covering 1995 to 2000. A selection of exemplary references had to be
made for this perspective overview.
Content: Data are included from studies on healthy humans,
gynecological patients, and persons suffering from inflammatory or
malignant diseases. The results indicate that competitive immunoassays
detect the total amount of circulating VEGF, which enables observations
regarding the increase in VEGF in pregnancy and preeclampsia to be
made. In these cases, capture immunoassays utilizing neutralizing
antibodies are insufficient because of an accompanying increase in
VEGF-binding soluble receptors (sFlt-1). Measurements of circulating
free VEGF are useful for study of malignant diseases, which are
associated with both genetically and hypoxia-induced overproduction of
VEGF. The VEGF isoform specificity of the antibodies is also critical
because both VEGF121 and VEGF165 are secreted.
It is important to consider that platelets and leukocytes release VEGF
during blood clotting.
Conclusions: Future efforts should concentrate on the balance
between free VEGF, total VEGF, and sFlt-1. Plasma, rather than serum,
should be used for analysis.
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Introduction
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Vascular endothelial growth factor
(VEGF)1
is a specific mitogen and survival factor for endothelial
cells and a key promoter of angiogenesis in physiological and
pathophysiological conditions (1)(2). VEGF is
required for the normal development of embryonic vasculature, the
cyclic growth of blood vessels in the female reproductive tract, and
the formation of capillaries during wound repair. Trials in
experimental animals and human patients have shown the therapeutic
potential of VEGF in coronary or peripheral arterial stenosis. However,
VEGF is also involved in abnormal angiogenesis, as seen in
proliferative retinopathies, rheumatoid arthritis, psoriasis, and
malignancies. In fact, VEGF plays a pivotal role in tumor expansion. It
locally initiates permeabilization of blood vessels, extravasation of
plasma proteins, invasion of stromal cells, and sprouting of new blood
vessels that supply the tumor with O2 and
nutriments and facilitate metastasis. Inhibition of angiogenesis is a
novel strategy in antitumor therapy (3)(4).
Initial studies revealed that the lungs, kidneys, heart, and adrenal
glands are the dominant sites of expression of the VEGF gene in
healthy adult animals (5). Today, it is assumed that
all tissues have the potential to produce the growth factor. Its
synthesis is stimulated when cells become deficient in
O2 or glucose and in inflammatory reactions.
Tumor cells tend to overexpress VEGF constitutively. VEGF acts
primarily in a paracrine way and binds to receptors of the basal
membranes of the endothelium. Hence, the question arises as to the
origin and function of blood-borne VEGF.
Approximately 300 publications dealing with measurements of circulating
VEGF for diagnostic and therapeutic monitoring have been published
during the past 6 years. However, understanding of the relationship
between the rate of the production of VEGF and its concentration in
blood is still insufficient. Several techniques for immunoassay of
circulating VEGF have been described. If one takes a glance at the
results, it becomes obvious that the data vary by up to three orders of
magnitude depending on the test applied. This review describes possible
reasons for these discrepancies.
Some investigators have used competitive immunoassays, which detect the
total amount of circulating VEGF, whereas others have used capture
immunoassays with neutralizing antibodies, which detect only free VEGF.
In addition, some assays have used antibodies that are specific for
single VEGF isoforms. Finally, recent studies have to be taken into
account that show that significant amounts of VEGF can be released from
platelets and leukocytes during blood sampling and handling.
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Molecular Biology of VEGF
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The human VEGF gene consists of eight exons and seven introns.
Transcriptional activation is mediated by binding of the trans-acting
dimeric protein hypoxia-inducible factor-1 (HIF-1
/ß) to a hypoxia
response element in the human VEGF gene promoter. The HIF-1
subunit is unstable in normoxia because it possesses a
PO2-dependent
degradation domain that targets it for ubiquitination. In addition,
VEGF mRNA is stabilized in hypoxia. Several proinflammatory cytokines,
such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor
(TNF-
), stimulate VEGF gene expression in a tissue-specific way
(2)(4). Recent evidence suggests that the
actions of IL-1 and TNF-
are also mediated through increased HIF-1
binding to DNA (6). The molecular mechanisms of the increase
in VEGF mRNA and VEGF protein production in response to glucose
deprivation are not yet understood. Hormones reported to influence VEGF
mRNA production include insulin, insulin-like growth factor-1,
corticotropin, thyrotropin, and steroidal hormones (7).
At least five isoforms of the protein, composed of 121, 145, 165, 189,
and 206 amino acids, can be translated because of alternative VEGF mRNA
splicing (2)(4). Glycosylation is essential for
efficient secretion. VEGF121 is a
freely soluble protein that does not bind heparin.
VEGF165, the predominant isoform, is a
heparin-binding basic homodimer of 45 kDa that remains partly bound to
the cell surface and the extracellular matrix. The other isoforms do
not enter the circulation in significant amounts because they are
either bound to the extracellular matrix
(VEGF145) or are secreted sparingly
(VEGF189 and VEGF206).
VEGF binds with high affinity to two tyrosine kinase receptors, the
fms-like tyrosine kinase (Flt-1, VEGFR-1) and the kinase domain
receptor (KDR, VEGFR-2), which are produced predominantly by
endothelial cells. Flt-1 is also present on trophoblasts and
macrophages, whereas KDR is present on hemopoietic stem cells,
megakaryocytes, and retinal cells. The production of Flt-1 and KDR
increases in response to hypoxia, although this increase is smaller
than that of VEGF. Binding of VEGF causes receptor dimerization and
autophosphorylation for signaling. The antiapoptotic and mitotic
functions of VEGF are mediated by KDR. VEGF165
can also bind to neuropilin-type receptors, which may explain
why VEGF165 is a more potent mitogen than
VEGF121. A detailed description of the structures
and functions of the various VEGF receptors has been provided by
Neufeld et al. (4).
The VEGF family of growth factors includes several related molecules,
such as placenta growth factor, VEGF-B, VEGF-C, VEGF-D, and
others. VEGF (VEGF-A) and its analogs have homologous amino acid
sequences and bind to tyrosine kinase receptors of the same class
(8)(9). This review is restricted to the
measurement of VEGF.
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Methods for Assaying Circulating VEGF
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Cell proliferation tests, receptor binding assays, or immunoassays
can be applied for VEGF quantification. VEGF (>100 ng/L)
stimulates the growth of endothelial cells in vitro. Keyt et al.
(10) demonstrated that response curves with glycosylated vs
nonglycosylated recombinant VEGF isoforms are identical. However,
endothelial cell proliferation tests are insufficient for assay of
circulating VEGF. Immunoassays are preferred in clinical practice,
although they may detect VEGF epitopes, even when the molecule is
devoid of biological activity. In-house RIAs with radiolabeled
VEGF (11), radioimmunometric assays with
radiolabeled monoclonal anti-VEGF antibody (12),
and immunochemiluminescence or ELISAs with either polyclonal
(13)(14) or monoclonal (15)
antibodies or a combination of these (16) have been
developed. The primary capture antibody can be replaced by recombinant
VEGF receptor molecules for ELISA (17). In addition,
commercial methods are available. Compared with bioassays,
immunoassays are characterized by low detection limits and greater
specificity, reproducibility, and practicability (18).
An international standard preparation of VEGF has not been established.
Comparative studies with different recombinant DNA-derived VEGF
products have not been carried out with respect to antibody binding
affinity and parallelism of dilution curves in immunoassays. The
importance of standardization of calibrants has been demonstrated in a
WHO study revealing substantial interassay differences in the results
obtained with commercial methods for IL-2, IL-6, and TNF-
(19).
Regarding the measurement of circulating VEGF, some assays detect only
VEGF121 (13) or only
VEGF165 (15), whereas others measure
the sum (VEGF121/165) of these
(15)(16)(17). A more crucial point is that capture ELISAs, with
recombinant VEGF receptors or neutralizing monoclonal antibodies,
selectively detect free VEGF. It remains to be investigated whether
changes in the concentration of free VEGF truly reflect VEGF
production, relative to degradation rates, or altered binding to
carrier proteins alone. A major potential VEGF-binding plasma protein
is
2-macroglobulin, which prevents the growth
factor from binding to its receptor (20). However, several
investigators have shown that
2-macroglobulin
does not interfere in their assay systems
(11)(16). Thus, it is unlikely that
2-macroglobulin is the main binding protein
masking VEGF in immunoassays.
In addition, the soluble form of VEGFR-1, sFlt-1, interacts with
circulating VEGF (17)(21)(22). Banks
et al. (23) partially purified and sequenced the
VEGF-binding activity in plasma samples from pregnant women and
demonstrated a novel multimeric receptor complex of 400550 kDa that
bound several VEGF molecules. Sandwich ELISAs with monoclonal
antibodies fail to detect the antigen if the epitopes are masked by
soluble receptors. Such interference has been described previously with
respect to measurements of circulating IL-1, IL-2, IL-6, and TNF-
(24). The common observation that the plasma concentrations
of soluble receptors for cytokines are high (10100 µg/L)
holds true for sFlt-1 (25). The total concentration of VEGF
(14) can be determined by competitive binding assays, i.e.,
by RIAs or fluorometric ELISAs that require only one epitope located in
a region of the molecule that is not occupied by a receptor molecule.
Interaction between VEGF and sFlt-1 must also be considered in assays
of tissue culture medium from cell lines expressing VEGF receptors
(26).
Assays have been marketed for the measurement of total VEGF (detection
limits
200 ng/L; Cytokit RedTM VEGF, CYTimmune
Sciences; ACCUCYTE®, Peninsula Laboratories) or
free VEGF121/165 (detection limits
10 ng/L;
Quantikine®, R&D Systems;
CYTELISATM, Peninsula Laboratories; hVEGF ELISA,
BioSource International). In the competitive binding assay reagent
sets for total VEGF, ELISA plates usually are coated with goat
anti-rabbit antibodies for capture of polyclonal rabbit anti-human VEGF
antibody. VEGF calibrators and samples are then added in a
competition reaction with biotinylated human VEGF. Commercial capture
ELISA methods for free VEGF use the sandwich technique, in which
monoclonal antibody specific for VEGF is precoated onto the plates.
After VEGF binding to the immobilized antibody, the enzyme-linked
second polyclonal or monoclonal antibody and substrate are added for
color development.
Faced with these substantial differences in the assay methods (Table 1
), it is not surprising that great variations exist when
published concentrations of circulating VEGF in healthy human subjects
are compared. Measured total VEGF concentrations of 325 µg/L have
been reported for competitive ELISAs (27)(28)(29), whereas
measured concentrations of
1 µg/L have been reported for RIAs
(11). The mean concentrations of free
VEGF121 and VEGF165 have
been reported as 19 ng/L (13) and 42 ng/L (15),
respectively. All of these values are independent of gender. In studies
incorporating the most commonly used commercial ELISA (Quantikine),
which detects the free isoforms VEGF121 and
VEGF165, plasma values of <9150 ng/L in
healthy subjects have been reported
(15)(23)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Higher values have
been measured by in-house assays with polyclonal antibodies for VEGF
(14). Furthermore, compared with plasma, the reference
interval for serum VEGF121/165 is relatively
high, averaging 10300 ng/L
(12)(15)(31)(35)(38)(41)(42).
The differences between plasma vs serum concentrations have been
ascribed to the release of VEGF from platelets and other blood cells
during clotting. On closer inspection, serum VEGF concentrations
reflect blood platelet counts rather than VEGF synthesis by peripheral
tissues (17)(30)(31). The serum VEGF
concentration further increases with clotting duration and temperature
(17). In addition to platelets, leukocytes can also secrete
VEGF (35)(43). Separate measurements of free
VEGF121/165 in blood cells (445 ng/L) and plasma
(19 ng/L) have underscored the relevance of blood cell-derived VEGF in
serum samples.
Citrated, EDTA-treated, or heparinized plasma processed in glass tubes
is the material of choice for measurement of VEGF. Plasma should be
frozen (-80 °C) within 1 h after venipuncture (31).
Alternatively, blood may be collected in CTAD tubes, which contain
citrate, theophylline, adenosine, and dipyridamole for platelet
stabilization (44). In the following discussion, references
will be restricted to measurements of VEGF in plasma, rather than
serum, whenever possible.
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Circulating VEGF in Pregnancy
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During pregnancy VEGF is essential for the proliferation of
trophoblasts, the development of embryonic vasculature, and the growth
of both maternal and fetal blood vessels in the uterus. Using a
competitive RIA, Anthony et al. (11) and Evans et al.
(45) demonstrated that maternal serum VEGF increases during
the first trimester of pregnancy (to 2.1 µg/L compared with 1.1
µg/L in nonpregnant women). Capture ELISAs with neutralizing
antibodies neither detect this increase, which is attributable to
sFlt-1 produced by the placenta (22)(23), nor
can they recover VEGF added to pregnancy samples
(11). Measurements of total VEGF in EDTA plasma by
nonradioactive competitive immunoassays yielded results of 12 µg/L in
normal pregnancies antepartum and 33 µg/L in gestational age-matched
patients with preeclampsia (46). Other investigators have
reported similar results (47), which support earlier
evidence obtained by a polyclonal antibody-based capture ELISA in serum
samples (48). Placental VEGF overproduction in response to
local hypoxia and inflammatory cytokines is involved in the etiology of
preeclampsia, which complicates 510% of all pregnancies. An
additional observation of diagnostic value is the increase in
circulating free VEGF after administration of human chorionic
gonadotropin to patients at risk from ovarian hyperstimulation
syndrome (29)(49)(50). Measurements
of total serum VEGF produced similar results in one study
(27), but not in another (29).
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Circulating VEGF in Response to Hypoxia and Inflammation
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Maloney et al. (51) found that the concentration of
free VEGF121/165 is not increased in the plasma
of mountaineers at extreme altitudes (14 200 feet) in association with
hypoxia or acute mountain sickness. Accordingly, the increased
serum VEGF concentrations measured in athletes training at high
altitudes have been related to activation of the immune system rather
than to hypoxic stress (52). Acute tissue hypoxia caused by
cigarette smoking is not a major stimulus for increased plasma free
VEGF121/165 concentrations (36).
However, the increased VEGF concentrations in serum from the superior
vena cava and the systemic arteries of children with cyanotic
congenital heart disease (53) could indicate local
stimulation of VEGF synthesis in response to systemic hypoxia.
Ischemia of the heart produces an acute increase in serum free
VEGF121/165 concentrations (42).
Administration of heparin to patients with acute myocardial infarction
rapidly lowers VEGF values (54). Disturbances of the
peripheral microcirculation can also lead to increased concentrations
of circulating VEGF as demonstrated in patients with chronic venous
disease (37) or sickle cell anemia (34). Whether
the increased concentrations of circulating free VEGF seen in diabetic
patients (25)(36)(55) are
attributable to peripheral hypoxia in association with angiopathies or
to impaired glucose metabolism remains to be investigated. Importantly,
Lip et al. (25) reported a significant decrease in plasma
free VEGF after successful laser treatment in patients with
proliferative retinopathy secondary to diabetes or ischemic retinal
vein occlusion.
VEGF promotes inflammatory processes by causing vascular leakage and
mobilizing leukocytes. Increased concentrations of free VEGF have been
measured in a variety of autoimmune and infectious inflammatory
diseases, including rheumatoid arthritis (56), POEMS
syndrome (57), and Kawasaki disease (58). This
increase may be produced not only by VEGF release from leukocytes and
platelets in circulation but also by exudation of the cytokine into the
circulation from inflamed organs.
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VEGF in Malignancies
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Angiogenesis is controlled by a fine local balance between
activating and inhibiting mediators (3). Increased
production of VEGF mRNA and synthesis of VEGF protein are
critical in tumor angiogenesis. Tumor cell-specific genetic alterations
lead to VEGF overproduction, even under normoxic conditions. On the
basis of ELISA measurements with impure VEGF calibrators and polyclonal
antibodies, Kondo et al. (59) first recognized the potential
of VEGF as a serum diagnostic marker for malignant diseases. Increased
serum concentrations of free VEGF have indeed been measured in various
types of cancer, including brain, lung, gastrointestinal,
hepatobiliary, renal, ovarian, and others. However, today it is clear
that VEGF found in serum is, to a large extent, released from platelets
during blood clotting (30)(31). Because blood
platelets in tumor patients contain more releasable VEGF than platelets
from healthy persons, Lee et al. (60) have argued that serum
is more useful than plasma in the diagnosis and follow-up of
malignancies. However, it is almost impossible to carry out
interlaboratory comparisons of VEGF serum data, mainly because the
procedures for blood handling are not standardized with respect to
clotting material, duration, and temperature. Therefore,
although we previously have shown that the concentration of free
VEGF121/165 is greatly increased in the serum of
patients with carcinomas or sarcomas and decreases after successful
chemotherapy (41), given the above problems the advice of
Banks et al. (31) to use plasma for assay is more accurate.
Careful reexamination using plasma samples has confirmed the concept
that the concentration of circulating free
VEGF121/165 is increased in malignant disease
(44)(61). Studies in patients with breast
(38), gastrointestinal (62), colorectal
(39), or prostate cancer (33) and melanoma
(40) have shown that plasma free
VEGF121/165 is increased further on development
of metastasis. Although values rarely exceeded 500 ng/L in these
studies, extremely high free VEGF121/165
concentrations (up to 463 µg/L) have been reported for patients with
leukemias or solid hematological tumors (63). A recent study
indicated that an angiogenic profile can be established for tumor
patients by measuring the plasma concentrations of the cytokines VEGF,
hepatocyte growth factor, basic fibroblast growth factor,
TNF-
, and transforming growth factor-ß. There is a regular
relationship between the concentrations of circulating VEGF and
hepatocyte growth factor and the extension of epithelial carcinomas.
Basic fibroblast growth factor concentrations usually are increased in
lung carcinoma, TNF-
concentrations in liver carcinoma, and both
cytokines in breast carcinomas (61). These cytokines may be
valuable diagnostic and prognostic markers at initial presentation and
during therapy of tumor patients.
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Perspectives
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VEGF is important in the local control of angiogenesis and
vascular permeability. Pharmacotherapeutic trials and genetic
engineering have already been performed in attempts to stimulate
VEGF-driven angiogenesis in vascular failure and to inhibit this
process in expanding tumors. However, several questions still remain
with respect to the role of VEGF as a circulating hormone. The plasma
concentration of free VEGF usually is very low in healthy
subjects. The low concentration of this growth factor
could be important in maintaining viability of the endothelium and
basic transport across the endothelial barrier. However, most VEGF
receptors are located on the basal membranes, thus rendering plasma
VEGF superfluous. There are two main stores for plasma VEGF. One
storage site is platelets, which take up VEGF and release it on
activation in vivo or in vitro. Therefore, serum is not recommended for
assay of VEGF. The other storage site is plasma proteins, namely
2-macroglobulin and sFlt-1, which bind VEGF.
Whether binding of VEGF to
2-macroglobulin is
a regulatory process still needs to be investigated. The VEGF-binding
capacity of the sFlt-1 fraction in plasma increases greatly during
pregnancy. The simultaneous increase in circulating VEGF is detectable
in competitive immunoassays but not in capture ELISAs with neutralizing
antibodies. Few reports are available concerning the measurement of
sFlt-1 and the total pool of VEGF in plasma. Intensive research is
required to improve understanding of the balance between free VEGF,
total VEGF, and its binding proteins. It seems likely that
2-macroglobulin and sFlt-1 target VEGF for
inactivation, although some hormones are protected from metabolism and
renal clearance by binding to carrier proteins. In malignancy and
inflammatory diseases, VEGF gene expression is greatly stimulated.
Here, plasma VEGF appears to escape from sFlt-1 binding. Genetically
determined overproduction of VEGF by tumor cells is thought to be more
important than hypoxia-induced VEGF gene expression, which is of
interest for therapeutic strategies to improve tumor oxygenation.
Measurement of plasma VEGF is expected to play an increasing role in
the diagnosis of patients suffering from malignancies and monitoring of
therapy.
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Acknowledgments
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I thank Dr. Bernhard F. Gibbs for linguistic improvement of the
manuscript. My studies are supported by the Deutsche
Forschungsgemeinschaft (SFB 367-C8).
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Footnotes
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1 Nonstandard abbreviations: VEGF, vascular endothelial growth factor; HIF-1, hypoxia-inducible factor-1; IL, interleukin; TNF-
, tumor necrosis factor
; Flt-1, fms-like tyrosine kinase; VEGFR, VEGF receptor; KDR, kinase domain receptor; and sFlt-1, soluble Flt-1. 
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