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Pitfalls in the Measurement of Circulating Vascular
Endothelial Growth Factor
Wolfgang Jelkmann
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, inflam-
matory diseases, and malignancies. However, there are
major quantitative differences in the published results.
This review attempts to identify reasons for these dis-
parities.
Approach: The literature was reviewed through a Med-
line search covering 1995 to 2000. A selection of exem-
plary 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 observa-
tions regarding the increase in VEGF in pregnancy and
preeclampsia to be made. In these cases, capture immu-
noassays utilizing neutralizing antibodies are insuffi-
cient 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 VEGF
121
and VEGF
165
are secreted. It is
important to consider that platelets and leukocytes re-
lease 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.
© 2001 American Association for Clinical Chemistry
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 patho-
physiological 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 in-
volved in abnormal angiogenesis, as seen in proliferative
retinopathies, rheumatoid arthritis, psoriasis, and malig-
nancies. 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 O
2
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 O
2
or glucose and in inflammatory
reactions. Tumor cells tend to overexpress VEGF consti-
tutively. VEGF acts primarily in a paracrine way and
binds to receptors of the basal membranes of the endo-
thelium. Hence, the question arises as to the origin and
function of blood-borne VEGF.
Approximately 300 publications dealing with measure-
Institut fu¨ r Physiologie, Medizinische Universita¨t zu Lu¨ beck, Ratzeburger
Allee 160, D-23538 Lu¨beck, Germany. Fax 49-451-500-4151; e-mail jelkmann@
physio.mu-luebeck.de.
Received July 31, 2000; accepted January 10, 2001.
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.
Clinical Chemistry 47:4
617–623 (2001)
Minireview
617
ments 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 immuno-
assay 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 immunoas-
says, 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.
Molecular Biology of VEGF
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 fac-
tor-1 (HIF-1
␣
/

) to a hypoxia response element in the
human VEGF gene promoter. The HIF-1
␣
subunit is
unstable in normoxia because it possesses a Po
2
-depen-
dent 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 bind-
ing to DNA (6 ). The molecular mechanisms of the in-
crease 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). Gly-
cosylation is essential for efficient secretion. VEGF
121
is
a freely soluble protein that does not bind heparin.
VEGF
165
, 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 extracellu-
lar matrix (VEGF
145
) or are secreted sparingly (VEGF
189
and VEGF
206
).
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, megakaryo-
cytes, 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 sig-
naling. The antiapoptotic and mitotic functions of VEGF
are mediated by KDR. VEGF
165
can also bind to neuropi-
lin-type receptors, which may explain why VEGF
165
is a
more potent mitogen than VEGF
121
. A detailed descrip-
tion 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.
Methods for Assaying Circulating VEGF
Cell proliferation tests, receptor binding assays, or immu-
noassays 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 prolifer-
ation 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 radio-
labeled VEGF (11), radioimmunometric assays with
radiolabeled monoclonal anti-VEGF antibody (12), and
immunochemiluminescence or ELISAs with either poly-
clonal (13, 14 ) or monoclonal (15 ) antibodies or a combi-
nation of these (16) have been developed. The primary
capture antibody can be replaced by recombinant VEGF
receptor molecules for ELISA (17 ). In addition, commer-
cial 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 re-
combinant 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 in-
terassay differences in the results obtained with commer-
cial methods for IL-2, IL-6, and TNF-
␣
(19).
Regarding the measurement of circulating VEGF, some
assays detect only VEGF
121
(13) or only VEGF
165
(15),
whereas others measure the sum (VEGF
121/165
) of these
(15–17). 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
618 Jelkmann: Measurement of Circulating VEGF
VEGF truly reflect VEGF production, relative to degrada-
tion rates, or altered binding to carrier proteins alone. A
major potential VEGF-binding plasma protein is
␣
2
-mac-
roglobulin, which prevents the growth factor from bind-
ing 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, inter-
acts with circulating VEGF (17, 21, 22). Banks et al. (23)
partially purified and sequenced the VEGF-binding activ-
ity in plasma samples from pregnant women and demon-
strated a novel multimeric receptor complex of 400 –550
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
(10–100
g/L) holds true for sFlt-1 (25 ). The total concen-
tration 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 con-
sidered 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 Red
TM
VEGF, CYTimmune Sciences; ACCUCYTE
®
, Peninsula
Laboratories) or free VEGF
121/165
(detection limits ⬃10
ng/L; Quantikine
®
, R&D Systems; CYTELISA
TM
, Penin-
sula Laboratories; hVEGF ELISA, BioSource Internation-
al). 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 mono-
clonal antibody specific for VEGF is precoated onto the
plates. After VEGF binding to the immobilized antibody,
the enzyme-linked second polyclonal or monoclonal an-
tibody 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 3–25
g/L have been reported for
competitive ELISAs (27–29), whereas measured concen-
trations of ⬃1
g/L have been reported for RIAs (11). The
mean concentrations of free VEGF
121
and VEGF
165
have
been reported as 19 ng/L (13) and 42 ng/L (15), respec-
tively. All of these values are independent of gender. In
studies incorporating the most commonly used commer-
cial ELISA (Quantikine), which detects the free isoforms
VEGF
121
and VEGF
165
, plasma values of ⬍9 –150 ng/L in
healthy subjects have been reported (15, 23, 30–40).
Higher values have been measured by in-house assays
with polyclonal antibodies for VEGF (14). Furthermore,
compared with plasma, the reference interval for serum
VEGF
121/165
is relatively high, averaging 10 –300 ng/L
(12, 15, 31, 35, 38, 41, 42).
The differences between plasma vs serum concentra-
tions 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 fur-
ther increases with clotting duration and temperature
(17). In addition to platelets, leukocytes can also secrete
VEGF (35, 43 ). Separate measurements of free VEGF
121/
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 pro-
cessed in glass tubes is the material of choice for measure-
ment 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, theophyl-
line, adenosine, and dipyridamole for platelet stabiliza-
tion (44 ). In the following discussion, references will be
restricted to measurements of VEGF in plasma, rather
than serum, whenever possible.
Circulating VEGF in Pregnancy
During pregnancy VEGF is essential for the proliferation
of trophoblasts, the development of embryonic vascula-
ture, 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 preg-
nancy (to 2.1
g/L compared with 1.1
g/L in nonpreg-
nant women). Capture ELISAs with neutralizing antibod-
ies 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 ). Mea-
surements of total VEGF in EDTA plasma by nonradio-
active competitive immunoassays yielded results of 12
g/L in normal pregnancies antepartum and 33
g/L in
Table 1. Characteristics of immunoassays for circulating
VEGF to be considered in interpretation of the results.
● Origin and type of calibrator
● VEGF isoform (VEGF
121
, VEGF
165
) specificity of antibodies
● Accuracy and quality of measurements of free or sFlt-1-bound
VEGF (neutralizing vs nonneutralizing monoclonal or polyclonal
antibodies)
● Interference with other VEGF-binding molecules (e.g.,
␣
2
-
macroglobulin or heparin)
● Type of sample (serum or plasma)
Clinical Chemistry 47, No. 4, 2001 619
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 hyp-
oxia and inflammatory cytokines is involved in the etiol-
ogy of preeclampsia, which complicates 5–10% of all
pregnancies. An additional observation of diagnostic
value is the increase in circulating free VEGF after admin-
istration 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 ).
Circulating VEGF in Response to Hypoxia and Inflammation
Maloney et al. (51) found that the concentration of free
VEGF
121/165
is not increased in the plasma of mountain-
eers at extreme altitudes (14 200 feet) in association with
hypoxia or acute mountain sickness. Accordingly, the
increased serum VEGF concentrations measured in ath-
letes 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
VEGF
121/165
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 stimula-
tion of VEGF synthesis in response to systemic hypoxia.
Ischemia of the heart produces an acute increase in
serum free VEGF
121/165
concentrations (42 ). Administra-
tion of heparin to patients with acute myocardial infarc-
tion 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 dis-
eases, including rheumatoid arthritis (56 ), POEMS syn-
drome (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.
VEGF in Malignancies
Angiogenesis is controlled by a fine local balance between
activating and inhibiting mediators (3 ). Increased produc-
tion 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 measure-
ments 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, re-
nal, 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 compari-
sons 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 concentra-
tion of free VEGF
121/165
is greatly increased in the serum
of patients with carcinomas or sarcomas and decreases
after successful chemotherapy (41), given the above prob-
lems the advice of Banks et al. (31 ) to use plasma for assay
is more accurate.
Careful reexamination using plasma samples has con-
firmed the concept that the concentration of circulating
free VEGF
121/165
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 VEGF
121/165
is increased
further on development of metastasis. Although values
rarely exceeded 500 ng/L in these studies, extremely high
free VEGF
121/165
concentrations (up to 463
g/L) have
been reported for patients with leukemias or solid hema-
tological 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-

. There is
a regular relationship between the concentrations of cir-
culating VEGF and hepatocyte growth factor and the
extension of epithelial carcinomas. Basic fibroblast growth
factor concentrations usually are increased in lung carci-
noma, 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.
Perspectives
VEGF is important in the local control of angiogenesis and
vascular permeability. Pharmacotherapeutic trials and
genetic engineering have already been performed in at-
tempts to stimulate VEGF-driven angiogenesis in vascular
failure and to inhibit this process in expanding tumors.
620 Jelkmann: Measurement of Circulating VEGF
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 endo-
thelium 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 detect-
able 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 re-
quired 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.
I thank Dr. Bernhard F. Gibbs for linguistic improvement
of the manuscript. My studies are supported by the
Deutsche Forschungsgemeinschaft (SFB 367-C8).
References
1. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability
factor/vascular endothelial growth factor, microvascular hyperper-
meability, and angiogenesis [Review]. Am J Pathol 1995;146:
1029 –39.
2. Ferrara N, Davis-Smyth T. The biology of vascular endothelial
growth factor [Review]. Endocr Rev 1997;18:4 –25.
3. Hanahan D, Folkman J. Patterns and emerging mechanisms of the
angiogenic switch during tumorigenesis [Review]. Cell 1996;86:
353– 64.
4. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endo-
thelial growth factor (VEGF) and its receptors [Review]. FASEB J
1999;13:9 –22.
5. Berse B, Brown LF, Van de Water L, Dvorak HF, Senger DR.
Vascular permeability factor (vascular endothelial growth factor)
gene is expressed differentially in normal tissues, macrophages,
and tumors. Mol Biol Cell 1992;3:211–20.
6. El Awad B, Kreft B, Wolber EM, Hellwig-Burgel T, Metzen E, Fandrey
J, Jelkmann W. Hypoxia and interleukin-1

stimulate vascular
endothelial growth factor production in human proximal tubular
cells. Kidney Int 2000;58:43–50.
7. Ferrara N. Role of vascular endothelial growth factor in the
regulation of angiogenesis [Review]. Kidney Int 1999;56:794–
814.
8. Nicosia RF. What is the role of vascular endothelial growth
factor-related molecules in tumor angiogenesis? [Review]. Am J
Pathol 1998;153:11– 6.
9. Joukov V, Kaipainen A, Jeltsch M, Pajusola K, Olofsson B, Kumar
V, et al. Vascular endothelial growth factors VEGF-B and VEGF-C
[Review]. J Cell Physiol 1997;173:211–5.
10. Keyt BA, Berleau LT, Nguyen HV, Chen H, Heinsohn H, Vandlen R,
Ferrara N. The carboxyl-terminal domain (111–165) of vascular
endothelial growth factor is critical for its mitogenic potency. J Biol
Chem 1996;271:7788 –95.
11. Anthony FW, Evans PW, Wheeler T, Wood PJ. Variation in detection
of VEGF in maternal serum by immunoassay and the possible
influence of binding proteins. Ann Clin Biochem 1997;34:276 –
80.
12. Waltenberger J, Lange J, Kranz A. Vascular endothelial growth
factor-A-induced chemotaxis of monocytes is attenuated in pa-
tients with diabetes mellitus. Circulation 2000;102:185–90.
13. Hanatani M, Tanaka Y, Kondo S, Ohmori I, Suzuki H. Sensitive
chemiluminescence enzyme immunoassay for vascular endothe-
lial growth factor/vascular permeability factor in human serum.
Biosci Biotechnol Biochem 1995;59:1958 –9.
14. Belgore F, Lip GY, Blann AD. Vascular endothelial growth factor
and its receptor, Flt-1, in smokers and non-smokers. Br J Biochem
Sci 2000;57:207–13.
15. Rodriguez CR, Fei DT, Keyt B, Baly DL. A sensitive fluorometric
enzyme-linked immunosorbent assay that measures vascular
endothelial growth factor 165 in human plasma. J Immunol
Methods 1998;219:45–55.
16. Schlaeppi JM, Eppenberger U, Martiny BG, Kung W. Chemilumi-
nescence immunoassay for vascular endothelial growth factor
(vascular permeability factor) in tumor-tissue homogenates. Clin
Chem 1996;42:1777– 84.
17. Webb NJ, Bottomley MJ, Watson CJ, Brenchley PE. Vascular
endothelial growth factor (VEGF) is released from platelets during
blood clotting: implications for measurement of circulating VEGF
levels in clinical disease. Clin Sci (Colch) 1998;94:395– 404.
18. Bienvenu JA, Monneret G, Gutowski MC, Fabien N. Cytokine
assays in human sera and tissues [Review]. Toxicology 1998;
129:55– 61.
19. Bienvenu J, Coulon L, Doche C, Gutowski MC, Grau GE. Analytical
performances of commercial ELISA-kits for IL-2, IL-6 and TNF-
␣
.A
WHO study. Eur Cytokine Netw 1993;4:447–51.
20. Soker S, Svahn CM, Neufeld G. Vascular endothelial growth factor
is inactivated by binding to
␣
2-macroglobulin and the binding is
inhibited by heparin. J Biol Chem 1993;268:7685–91.
21. Kendall RL, Wang G, Thomas KA. Identification of a natural
soluble form of the vascular endothelial growth factor receptor,
FLT-1, and its heterodimerization with KDR. Biochem Biophys Res
Commun 1996;226:324 – 8.
22. Clark DE, Smith SK, He Y, Day KA, Licence DR, Corps AN, et al. A
vascular endothelial growth factor antagonist is produced by the
human placenta and released into the maternal circulation. Biol
Reprod 1998;59:1540 – 8.
23. Banks RE, Forbes MA, Searles J, Pappin D, Canas B, Rahman D,
et al. Evidence for the existence of a novel pregnancy-associated
Clinical Chemistry 47, No. 4, 2001 621
soluble variant of the vascular endothelial growth factor receptor,
Flt-1. Mol Hum Reprod 1998;4:377– 86.
24. Radoux D, De Groote D. The total cytokine concept: the influence
of soluble receptors in the cytokine measurement. In: Bergmann
L, Mitrou PS, eds. Cytokines in cancer therapy. Basel: Karger,
1994:251–9.
25. Lip PL, Belgore F, Blann AD, Hope-Ross MW, Gibson JM, Lip GY.
Plasma VEGF and soluble VEGF receptor FLT-1 in proliferative
retinopathy: relationship to endothelial dysfunction and laser
treatment. Invest Ophthalmol Vis Sci 2000;41:2115–9.
26. Hornig C, Behn T, Bartsch W, Yayon A, Weich HA. Detection and
quantification of complexed and free soluble human vascular
endothelial growth factor receptor-1 (sVEGFR-1) by ELISA. J Immu-
nol Methods 1999;226:169 –77.
27. Agrawal R, Conway G, Sladkevicius P, Tan SL, Engmann L, Payne
N, et al. Serum vascular endothelial growth factor and Doppler
blood flow velocities in in vitro fertilization: relevance to ovarian
hyperstimulation syndrome and polycystic ovaries. Fertil Steril
1998;70:651– 8.
28. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH,
Sharkey AM, Smith SK. Vascular endothelial growth factor is
produced by peritoneal fluid macrophages in endometriosis and is
regulated by ovarian steroids. J Clin Invest 1996;98:482–9.
29. Ludwig M, Jelkmann W, Bauer O, Diedrich K. Prediction of severe
ovarian hyperstimulation syndrome by free serum vascular endo-
thelial growth factor concentration on the day of human chorionic
gonadotrophin administration. Hum Reprod 1999;14:2437– 41.
30. Verheul HM, Hoekman K, Luykx-de Bakker S, Eekman CA, Folman
CC, Broxterman HJ, Pinedo HM. Platelet: transporter of vascular
endothelial growth factor. Clin Cancer Res 1997;3:2187–90.
31. Banks RE, Forbes MA, Kinsey SE, Stanley A, Ingham E, Walters C,
Selby PJ. Release of the angiogenic cytokine vascular endothelial
growth factor (VEGF) from platelets: significance for VEGF mea-
surements and cancer biology. Br J Cancer 1998;77:956 – 64.
32. Maloney JP, Silliman CC, Ambruso DR, Wang J, Tuder RM, Voelkel
NF. In vitro release of vascular endothelial growth factor during
platelet aggregation. Am J Physiol 1998;275:H1054 – 61.
33. Duque JL, Loughlin KR, Adam RM, Kantoff PW, Zurakowski D,
Freeman MR. Plasma levels of vascular endothelial growth factor
are increased in patients with metastatic prostate cancer. Urology
1999;54:523–7.
34. Solovey A, Gui L, Ramakrishnan S, Steinberg MH, Hebbel RP.
Sickle cell anemia as a possible state of enhanced anti-apoptotic
tone: survival effect of vascular endothelial growth factor on
circulating and unanchored endothelial cells. Blood 1999;93:
3824 –30.
35. Nielsen HJ, Werther K, Mynster T, Brunner N. Soluble vascular
endothelial growth factor in various blood transfusion compo-
nents. Transfusion 1999;39:1078 – 83.
36. Wasada T, Kawahara R, Katsumori K, Naruse M, Omori Y. Plasma
concentration of immunoreactive vascular endothelial growth fac-
tor and its relation to smoking. Metabolism 1998;47:27–30.
37. Shoab SS, Scurr JH, Coleridge-Smith PD. Increased plasma
vascular endothelial growth factor among patients with chronic
venous disease. J Vasc Surg 1998;28:535– 40.
38. Adams J, Carder PJ, Downey S, Forbes MA, MacLennan K, Allgar
V, et al. Vascular endothelial growth factor (VEGF) in breast
cancer: comparison of plasma, serum, and tissue VEGF and
microvessel density and effects of tamoxifen. Cancer Res 2000;
60:2898 –905.
39. Davies MM, Jonas SK, Kaur S, Allen-Mersh TG. Plasma vascular
endothelial but not fibroblast growth factor levels correlate with
colorectal liver metastasis vascularity and volume. Br J Cancer
2000;82:1004 – 8.
40. Redondo P, Bandres E, Solano T, Okroujnov I, Garcia FJ. Vascular
endothelial growth factor (VEGF) and melanoma.
N
-Acetylcysteine
downregulates VEGF production in vitro. Cytokine 2000;12:
374–8.
41. Heits F, Katschinski DM, Wiedemann GJ, Weiss C, Jelkmann W.
Serum vascular endothelial growth factor (VEGF), a prognostic
indicator in sarcoma and carcinoma patients. Int J Oncol 1997;
10:333–7.
42. Seko Y, Imai Y, Suzuki S, Kamijukkoku S, Hayasaki K, Sakomura
Y, et al. Serum levels of vascular endothelial growth factor in
patients with acute myocardial infarction undergoing reperfusion
therapy. Clin Sci (Colch) 1997;92:453– 4.
43. Webb NJ, Myers CR, Watson CJ, Bottomley MJ, Brenchley PE.
Activated human neutrophils express vascular endothelial growth
factor (VEGF). Cytokine 1998;10:254 –7.
44. Wynendaele W, Derua R, Hoylaerts MF, Pawinski A, Waelkens E,
de Bruijn EA, et al. Vascular endothelial growth factor measured in
platelet poor plasma allows optimal separation between cancer
patients and volunteers: a key to study an angiogenic marker in
vivo? Ann Oncol 1999;10:965–71.
45. Evans P, Wheeler T, Anthony F, Osmond C. Maternal serum
vascular endothelial growth factor during early pregnancy. Clin Sci
(Colch) 1997;92:567–71.
46. Sharkey AM, Cooper JC, Balmforth JR, McLaren J, Clark DE,
Charnock-Jones DS, et al. Maternal plasma levels of vascular
endothelial growth factor in normotensive pregnancies and in
pregnancies complicated by pre-eclampsia. Eur J Clin Invest
1996;26:1182–5.
47. Kupferminc MJ, Daniel Y, Englender T, Baram A, Many A, Jaffa AJ,
et al. Vascular endothelial growth factor is increased in patients
with preeclampsia. Am J Reprod Immunol 1997;38:302– 6.
48. Baker PN, Krasnow J, Roberts JM, Yeo KT. Elevated serum levels
of vascular endothelial growth factor in patients with preeclamp-
sia. Obstet Gynecol 1995;86:815–21.
49. Artini PG, Fasciani A, Monti M, Luisi S, D’Ambrogio G, Genazzani
AR. Changes in vascular endothelial growth factor levels and the
risk of ovarian hyperstimulation syndrome in women enrolled in an
in vitro fertilization program. Fertil Steril 1998;70:560 – 4.
50. Abramov Y, Barak V, Nisman B, Schenker JG. Vascular endothelial
growth factor plasma levels correlate to the clinical picture in
severe ovarian hyperstimulation syndrome. Fertil Steril 1997;67:
261–5.
51. Maloney J, Wang D, Duncan T, Voelkel N, Ruoss S. Plasma
vascular endothelial growth factor in acute mountain sickness.
Chest 2000;118:47–52.
52. Schobersberger W, Hobisch-Hagen P, Fries P, Wiedermann F,
Rieder-Scharinger J, Herold M, et al. Increase in immune activa-
tion, vascular endothelial growth factor and erythropoietin after an
ultramarathon run at moderate altitude. Immunobiology 2000;
201:611–20.
53. Starnes SL, Duncan BW, Kneebone JM, Rosenthal GL, Jones TK,
Grifka RG, et al. Vascular endothelial growth factor and basic
fibroblast growth factor in children with cyanotic congenital heart
disease. J Thorac Cardiovasc Surg 2000;119:534 –9.
54. Kawamoto A, Kawata H, Akai Y, Katsuyama Y, Takase E, Sasaki Y,
et al. Serum levels of VEGF and basic FGF in the subacute phase
of myocardial infarction. Int J Cardiol 1998;67:47–54.
55. McLaren M, Elhadd TA, Greene SA, Belch JJ. Elevated plasma
vascular endothelial cell growth factor and thrombomodulin in
juvenile diabetic patients. Clin Appl Thromb Hemost 1999;5:
21– 4.
56. Bottomley MJ, Webb NJ, Watson CJ, Holt L, Bukhari M, Denton J,
et al. Placenta growth factor (PlGF) induces vascular endothelial
growth factor (VEGF) secretion from mononuclear cells and is
co-expressed with VEGF in synovial fluid. Clin Exp Immunol
2000;119:182– 8.
622 Jelkmann: Measurement of Circulating VEGF
57. Soubrier M, Dubost JJ, Serre AF, Ristori JM, Sauvezie B, Cathe-
bras P, et al. Growth factors in POEMS syndrome: evidence for a
marked increase in circulating vascular endothelial growth factor.
Arthritis Rheum 1997;40:786 –7.
58. Terai M, Yasukawa K, Narumoto S, Tateno S, Oana S, Kohno Y.
Vascular endothelial growth factor in acute Kawasaki disease.
Am J Cardiol 1999;83:337–9.
59. Kondo S, Asano M, Matsuo K, Ohmori I, Suzuki H. Vascular
endothelial growth factor/vascular permeability factor is detect-
able in the sera of tumor-bearing mice and cancer patients.
Biochim Biophys Acta 1994;1221:211– 4.
60. Lee JK, Hong YJ, Han CJ, Hwang DY, Hong SI. Clinical usefulness
of serum and plasma vascular endothelial growth factor in cancer
patients: which is the optimal specimen? Int J Oncol 2000;17:
149 –52.
61. Fuhrmann-Benzakein E, Ma MN, Rubbia-Brandt L, Mentha G,
Ruefenacht D, Sappino AP, Pepper MS. Elevated levels of angio-
genic cytokines in the plasma of cancer patients. Int J Cancer
2000;85:40 –5.
62. Yoshikawa T, Tsuburaya A, Kobayashi O, Sairenji M, Motohashi H,
Yanoma S, Noguchi Y. Plasma concentrations of VEGF and
bFGF in patients with gastric carcinoma. Cancer Lett 2000;153:
7–12.
63. Belgore F, Lip GY, Bareford D, Blann AD. Plasma levels of vascular
endothelial growth factor (VEGF) in haematological cancers [Let-
ter]. Br J Haematol 2000;110:496 –7.
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