Clinical Science (2005) 109, 39–43 (Printed in Great Britain) 39

Plasma vascular endothelial growth factor

as a marker for early vascular damage
in hypertension

Wei-Chuan TSAI∗ , Yi-Heng LI∗ , Yao-Yi HUANG†, Chin-Chan LIN†,

Ting-Hsing CHAO∗ and Jyh-Hong CHEN∗
∗
Department of Internal Medicine, National Cheng Kung University Medical Centre, Tainan, Taiwan, and
†Department of Emergency Medicine, National Cheng Kung University Medical Centre, Tainan, Taiwan

A B S T R A C T

Elevation of plasma VEGF (vascular endothelial growth factor) has been noted in patients with
hypertension or atherosclerosis. VEGF has been regarded as a marker for endothelial dysfunction.
However, the role of VEGF in hypertension-induced vascular injury and its relationship with endo-
thelial function have not been studied. This study included 20 untreated hypertensive men with
grade 1 or 2 hypertensive retinopathy, 10 untreated hypertensive men without hypertensive retino-
pathy and 10 healthy controls. None of the hypertensive patients had diabetes, renal impairment
or overt vascular diseases. Plasma VEGF and adhesion molecules were measured using ELISAs.
Endothelial function was measured by FMD (flow-mediated vasodilation) of the brachial artery.
Plasma levels of VEGF, excluding adhesion molecules, were significantly higher in hypertensive
patients with retinopathy when compared with patients without retinopathy (152.4 + − 80.8 pg/ml
versus 104.7 +− 27.2 pg/ml, P = 0.035) or controls (152.4 +
− 80.8 pg/ml versus 98.9 +
− 23.7 pg/ml, P =
0.025). Levels of FMD were significantly lower in hypertensive patients than controls, but
there were no significant differences between patients with or without retinopathy. Degrees of
FMD were inversely correlated with VEGF levels (r = − 0.351, P = 0.031). Elevation of plasma
VEGF was associated with hypertensive retinopathy. Plasma VEGF could be used as a marker of
early vascular damage induced by hypertension.

INTRODUCTION creased in acute heart failure [6], and could be a factor

VEGF (vascular endothelial growth factor) is known to
be a multifunctional peptide capable of inducing receptor-
mediated endothelial cell proliferation and angiogenesis
[1]. Recently, a link between VEGF and cardiovascular
diseases has been established. Elevation of VEGF has
been noted following acute myocardial infarction [2,3].
It has also been associated with coronary artery disease
and peripheral vascular disease [4], and has been noted
after coronary artery bypass surgery [5]. VEGF is also in-
for poor prognosis in acute coronary syndrome [7]. Even
patients with coronary risk factors, such as hyperlipid-
aemia [8] and hypertension [9], showed association with
increased VEGF. Impaired regulation of vascular growth
has been noted in hypertension [10]. Previous studies
have demonstrated that VEGF and its soluble receptor
Flt-1 increase in hypertension and can be reduced after
antihypertensive treatment [9]. However, the importance
of VEGF in the development of vascular damage in asso-
ciation with hypertension has not been fully elucidated.

Key words: adhesion molecule, flow-mediated vasodilation (FMD), hypertension, retinopathy, vascular damage, vascular endothelial
growth factor (VEGF).
Abbreviations: FMD, flow-mediated vasodilation; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion
molecule 1; VEGF, vascular endothelial growth factor.
Correspondence: Dr Wei-Chuan Tsai (email wctsai@ksmail.seed.net.tw).


C 2005 The Biochemical Society
40 W.-C. Tsai and others
Increased expression of adhesion molecules, such as
ICAM-1 (intercellular adhesion molecule 1) and VCAM-
1 (vascular cell adhesion molecule 1), has been noted when
the endothelium is activated by inflammatory response
[11]. Soluble ICAM-1 and VCAM-1 were increased in
hypertension [12]. We hypothesized that VEGF and ad-
hesion molecules could be early markers for the develop-
ment of vascular damage in hypertension. As hyper-
tensive retinal microvascular signs are important markers
of microvascular damage of other organs due to elevated
blood pressure [13,14], we used early changes in hyper-
tensive retinopathy as an index of early vascular damage
in hypertension and studied this hypothesis in the present
study.
VEGF has also been regarded as an endothelial marker.
Increased plasma VEGF concentration might simply ref-
lect endothelial damage caused by hypertension [1]. In
order to investigate whether VEGF was only an endo-
thelial damage marker in the development of vascular
damage in hypertension, we also measured FMD (flow-
mediated vasodilation) in each subject to observe the
relationship between VEGF and endothelial function.
EXPERIMENTAL
Subjects
Twenty untreated hypertensive men (age, 38.8 + − Measurement of adhesion molecules
9.8 years) with grade 1 or 2 hypertensive retinopathy, 10
untreated hypertensive men (age, 31.7 + − 9.8 years) with-
out retinopathy and 10 age-matched healthy men (age
32.0 +
− 7.4 years) as controls were recruited for the present
study. All patients were recruited from a hypertensive
clinic. They had not been receiving any antihyper-
tensive medication prior to recruitment. Blood pressure
was measured using the standard sphygmomanometry
method used in clinic. Hypertension was diagnosed if
blood pressure > 140/90 mmHg on two separate oc-
casions. None of the patients had other overt vascular
diseases. Controls were recruited from healthy volunteers
from our previous study [17], after careful evaluation
for the risk factors. They were all non-smokers and did
not show any risk factors. None of the subjects drank
alcohol. Subjects with a fasting blood sugar more than
110 mg/dl or a body mass index greater than 25 kg/m2
were excluded. For the retinopathy evaluation, direct
or indirect ophthalmoscopy was performed on all hyper-
tensive subjects after dilatation of the pupils. In order
to reduce the possibility of misclassification, the fundo-
scopic examination was performed by a retinopathy
specialist, who was a blind observer. The grade of hyper-
tensive retinopathy was determined according to the
Keith–Wagener classification [15]. Grade 1 (narrowing
of the vessels) and grade 2 (pressure from the artery on
the vein at arteriovenous crossings) retinopathies were
regarded as early vascular damage.

C 2005 The Biochemical Society
Written informed consent was obtained from all of
the subjects, and the study was approved by the Clinical
Research Committee of the hospital.
Measurement of plasma VEGF
Fasting blood samples were drawn from the antecubital
vein and the samples were treated with the anticoagulant
trisodium citrate. All the blood samples were taken within
1 week after recruitment. After centrifugation at 1000 g
for 20 minutes, the plasma was immediately separated and
frozen to − 70 ◦ C until examination. Plasma concentra-
tions of VEGF were assayed by ELISA (R&D Systems,
Minneapolis, MN, U.S.A.). In brief, a monoclonal anti-
body specific for VEGF was pre-coated on to a micro-
plate. Standards and samples were pipetted into the wells,
and any VEGF present was bound by the immobilized
antibody. After washing away any unbound sub-
stances, an enzyme-linked polyclonal antibody specifi-
cally against VEGF was added to the wells. Following a
wash to remove any unbound antibody–enzyme reagent,
a substrate solution was added to the wells and the colour
developed in proportion to the amount of VEGF bound
in the initial step. Colour development was then stopped,
and the intensity of the colour was measured using a
microplate reader set at A450 . The minimum detectable
amount of VEGF was 5.0 pg/ml.
Plasma concentrations of soluble ICAM-1 and VCAM-1
were measured by ELISAs (R&D Systems), as described
previously [16]. In brief, a monoclonal antibody specific
to ICAM-1 or VCAM-1 was pre-coated on to a micro-
plate. Standards, samples, controls and conjugate were
pipetted into the wells and any ICAM-1 or VCAM-1
present was sandwiched by the immobilized antibody and
the enzyme-linked monoclonal antibody specifically
against ICAM-1 or VCAM-1. Following a wash to re-
move any unbound substances or antibody–enzyme
reagents, a substrate solution was added to the wells and
the colour developed in proportion to the amount of
ICAM-1 or VCAM-1 bound. The colour development
was stopped, and the intensity of the colour was mea-
sured. The minimum detectable dose was 0.35 ug/l for
ICAM-1 and 2.0 ug/l for VCAM-1.
Measurement of FMD
FMD of the brachial artery was measured in a quiet,
temperature-controlled room after 10 min of bed rest
using a method described previously [17]. Briefly, FMD,
in response to reactive hyperaemia, was measured in
the left brachial artery. A high-resolution ultrasound
machine (Hewlett-Packard Sonos 2500) equipped with
a 7.5-MHz linear array probe was used for the present
study. Arterial diameters were measured at the baseline
and during reactive hyperaemia. The condition of reactive
hyperaemia was induced by inflation of a pneumatic cuff
 Vascular endothelial growth factor and hypertension 41

Table 1 Basic data and measurements between groups

The results are expressed as the means + ∗
− S.D. or number of patients (%); P < 0.05. SBP, systolic blood pressure; DBP, diastolic blood pressure.
Parameter Hypertensives with retinopathy (n = 20) Hypertensives without retinopathy (n = 10) Controls (n = 10)

Age (years) 38.8 +

− 9.8 31.7 +
− 9.8 32.0 +
− 7.4
Body mass index (kg/m2 ) 23.1 +
− 1.0 23.1 +
− 1.1 22.8 +
− 1.1
SBP (mmHg) 182.4 +
− 35.9 166.0 +
− 28.7 119.8 +
− 12.4
∗

DBP (mmHg) 120.0 +

− 23.0 112.3 +
− 21.5 74.0 +
− 6.6
∗

Heart rate (beats/min) 75.5 +

− 8.5 71.5 +
− 7.4 69.8 +
− 7.4
Cholesterol (mg/dl) 201.4 +
− 31.8 176.1 +
− 27.7
∗
214.4 +
− 37.0
Triacylglycerol (mg/dl) 152.8 +
− 73.7 171.4 +
− 83.1 147.9 +
− 65.6
Fasting blood glucose (mg/dl) 88.0 +
− 6.6 84.0 +
− 3.5 85.4 +
− 9.8
Serum creatinine (mg/dl) 1.0 +
− 0.1 0.9 +
− 0.1 0.9 +
− 0.1
VEGF (pg/ml) 152.4 +
− 80.8
∗
104.7 +
− 27.2 98.9 +
− 23.7
VCAM-1 (µg/l) 416.1 +
− 76.3 384.1 +
− 48.7 400.0 +
− 92.5
ICAM-1 (µg/l) 198.1 +
− 43.7 184.0 +
− 29.7 193.4 +
− 44.7
FMD (%) 4.4 +
− 1.7 5.1 +
− 1.6 9.6 +
− 2.1
∗

Smoking 6 (30 %) 4 (40 %) 0

on the forearm to a pressure above 250 mmHg for 4.5 min.

The brachial artery was scanned in longitudinal sections
2 to 5 cm above the elbow. The arterial diameter was
measured on B-mode images at the end-diastolic phase
from one media–adventitia interface to the other at the
clearest section for six times at baseline. The average of
the six measurements was considered as the baseline data.
We then measured three more times every 30 s after
reactive hyperaemia. The average of the three consecutive
maximal diameters was taken as the data after hyperaemia.
FMD was calculated as the percentage change in diameter
compared with the baseline. In our laboratory, two
independent investigators performed the measurements.
The intra-observer and inter-observer variations were
0.9 % and 1.4 % respectively. The true variability of the
method was about 15–20 % in the present study.

Statistics Figure 1 Levels of VEGF were significantly higher in

The results are expressed as the means +
− S.D. A Kruskal–
Wallis statistical analysis followed by a Mann–Whitney
rank-sum test were used for comparison of continuous
variables between groups. A Spearman’s rank correlation
test was used for assessment of the relation between
FMD, VEGF and adhesion molecules. A P value < 0.05
was considered statistically significant. Analysis was
performed using SPSS version 10.0 for Windows (SPSS,
Chicago, IL, U.S.A.).
RESULTS
Table 1 shows the clinical characteristics and measure-
ments in hypertensive patients with and without retino-
pathy and in controls. Blood pressure was significantly
higher in hypertensive patients than controls, although
hypertensive patients with retinopathy than in hypertensive
patients without retinopathy or controls
there was no difference between patients with or
without retinopathy. Current smoking status was simi-
lar in the two groups of hypertension. Cholesterol
was significantly lower in hypertensive patients without
retinopathy than in the other two groups. Plasma levels
of VEGF, but not adhesion molecules, were significantly
higher in hypertensive patients with retinopathy when
compared with patients without retinopathy or controls.
There were no differences in VEGF levels between
grade 1 (n = 16) and grade 2 (n = 4) retinopathy. There
were no significant differences in VEGF levels between
patients without retinopathy and controls (Figure 1).
Levels of FMD were significantly lower in hypertensive
patients than in controls, but there were no significant


C 2005 The Biochemical Society
42 W.-C. Tsai and others

be used as a marker for early microvascular damage in

hypertension. Previous studies have demonstrated that
VEGF is increased in hypertensive patients and decreased
after control of blood pressure [9]. In our study, elevation
of VEGF was noted among hypertensive patients only
when vascular damage occurred in the retina. VEGF can
be a marker for early microvascular damage in hyper-
tension. Previous studies have shown that adhesion mol-
ecules are elevated in elderly hypertension [12]. Levels
of VCAM-1 and ICAM-1 are associated with age and
blood pressure [12]. However, adhesion molecules were
not elevated in hypertensive patients in our study. This
discrepancy is probably due to the fact that our patients
were relatively young and in the early stage of hyper-
tension. Adhesion molecules were not good markers for
vascular damage in the early stage of hypertension. The
relationship between blood pressure and adhesion mol-
Figure 2 Degrees of FMD were significantly lower in ecules has also been inconsistent in previous studies [11].
hypertensive patients than in controls There are several proposed mechanisms responsible for
the elevation of VEGF in vascular diseases. First, in re-
Table 2 Influences of age, blood pressure and lipid profiles sponse to vascular damage, a wide array of growth factors,
on VEGF and FMD cytokines and other molecules are released, stimulating
SBP, systolic blood pressure; DBP, diastolic blood pressure. angiogenesis via VEGF, which is essential for the repair
VEGF FMD process [1]. Another possible mechanism is that elevation
of VEGF may simply reflect endothelial cell damage
r P r P apparent in hypertension [1]. In our study, FMD in hyper-
tensive patients was significantly lower than in normal
Age 0.091 0.588 − 0.258 0.108
controls. It reflected macrovascular dysfunction in hyper-
SBP 0.223 0.179 − 0.516 0.001
tension. The degrees of FMD were negatively correlated
DBP 0.228 0.169 − 0.550 < 0.001
with the levels of VEGF. However, elevation of VEGF
Cholesterol 0.126 0.457 0.172 0.302
could only partially reflect endothelial cell damage caused
Triacylglycerol − 0.016 0.925 0.049 0.771
by hypertension, since VEGF did not elevate in hyper-
tensive patients without retinopathy. Association of
VEGF and microvascular damage is possibly through
differences between patients with or without retinopathy
other mechanisms. The fact that elevated VEGF is not
(Figure 2). Degrees of FMD were inversely correlated
solely related to endothelial damage has been supported
with VEGF levels (r = − 0.351, P = 0.031) in all subjects
by the lack of a correlation between VEGF and the von
pooled. However, VEGF and FMD were not correlated
Willebrand factor [8].
with levels of adhesion molecules. VEGF was not
Similar findings have also been reported in diabetes pa-
correlated with blood pressure, lipid profiles or age.
tients [4]. VEGF has been found to be raised only in dia-
FMD was significantly correlated with blood pressure,
betic patients with vascular disease, rather than diabetes
but not lipid profiles or age (Table 2). Smoking status
alone. Elevation of VEGF is associated with athero-
did not influence the levels of VEGF and FMD. After
sclerosis, but not diabetes itself [4]. A positive correlation
multivariate regression analysis controlling for age, blood
between coronary artery collaterals and intra-coronary
pressure, smoking status, cholesterol and fasting
artery VEGF has been reported [18]. All of the evidence,
blood sugar, VEGF levels were still significantly different
as well as our observations, support the finding that
between patients with or without retinopathy (P = 0.013).
elevation of VEGF levels is associated with vascular
atherosclerosis caused by various risk factors.
DISCUSSION The limitation of this study was that the retinal changes
were not specific for hypertension. There was a trend for
The present study showed that VEGF was significantly patients with retinopathy to be older and with higher
higher in hypertensive patients with retinopathy than blood pressure and cholesterol. These factors could have
in hypertensive patients without retinopathy or normo- confounding effects on our results. The cumulative effects
tensive subjects. There were no significant differences of age, smoking, cholesterol levels and fasting blood
in adhesion molecules between the groups. This result sugar probably contributed to atherosclerosis in the
indicates that VEGF, rather than adhesion molecules, can retinopathy group independent of hypertensive vascular


C 2005 The Biochemical Society

changes. Age, blood pressure levels and cholesterol did
not correlated with VEGF levels, and VEGF levels were
still significantly different between patients with or with-
out retinopathy after multivariable analysis controlling
for age, blood pressure, smoking status, cholesterol and
blood sugar. The effects of these factors in our study
were probably small, and a large-scale study is needed
for further investigation. The other limitation of our
study was that we did not obtain urine for measurement
of microalbumin. Microalbuminuria is known to be a
reliable and simple way to detect endothelial damage.
In conclusion, our study demonstrates that plasma
VEGF levels can be a useful marker for the detection of
early microvascular damage in hypertension. Decreased
levels of VEGF have been noted after treatment for hyper-
tension or hyperlipidaemia [8,9]. However, there has
been no direct evidence to indicate that decreased VEGF
is associated with reduced atherosclerosis. The role of
VEGF in the long-term prognosis for cardiovascular
events in hypertension is worth further evaluation.
ACKNOWLEDGMENTS
This study was supported by the MOE Program for Pro-
moting Academic Excellence of Universities under grant
number 91-B-FA09-2-4 and by grants NSC89-2314-B-
006-180 and NSC90-2314-B-006-085 from the National
Science Council, Executive Yuan, Taipei, Taiwan.
REFERENCES
1 Felmeden, D. C., Blann, A. D. and Lip, G. Y. H. (2003)
Angiogenesis: basic pathophysiology and implications for
disease. Eur. Heart J. 24, 586–603
2 Seko, Y., Imai, Y., Suzuki, S. et al. (1997) Serum levels of
vascular endothelial growth factor in patients with acute
myocardial infarction undergoing reperfusion therapy.
Clin. Sci. 92, 453–454
3 Hojo, Y., Ikeda, U., Zhu, Y. et al. (2000) Expression of
vascular endothelial growth factor in patients with acute
myocardial infarction. J. Am. Coll. Cardiol. 35, 968–973
4 Blann, A. D., Belgore, F. M., McCollum, C. N.,
Silverman, S., Lip, P. L. and Lip, G. Y. H. (2002) Vascular
endothelial growth factor and its receptor Flt-1 in the
plasma of patients with coronary and peripheral
atherosclerosis and type II diabetes. Clin. Sci. 102, 187–194
Received 21 October 2004/24 January 2005; accepted 1 March 2005
Published as Immediate Publication 1 March 2005, DOI 10.1042/CS20040307
Vascular endothelial growth factor and hypertension 43
5 Burton, P. B. J., Owen, V. J., Hafizi, S. et al. (2000) Vascular
endothelial growth factor release following coronary artery
bypass surgery: extracorporeal circulation versus ‘beating
heart’ surgery. Eur. Heart J. 21, 1708–1713
6 Chin, B. S. P., Chung, N. A., Gibbs, C. R., Blann, A. D.
and Lip, G. Y. H. (2002) Vascular endothelial growth
factor and soluble P-selectin in acute and chronic
congestive heart failure. Am. J. Cardiol. 90, 1258–1260
7 Heeschen, C., Dimmeler, S., Hamm, C. W., Boersma, E.,
Zeiher, A. M. and Simoons, M. L. (2003) Prognostic
significance of angiogenic growth factor serum levels in
patients with acute coronary syndrome. Circulation 107,
524–530
8 Blann, A. D., Belgore, F. M., Constans, J., Conri, C. and
Lip, G. Y. H. (2001) Plasma vascular endothelial growth
factor and its receptor Flt-1 in patients with
hyperlipidemia and atherosclerosis and the effects
of fluvastatin or fenofibrate. Am. J. Cardiol. 87,
1160–1163
9 Belgore, F. M., Blann, A. D., Li-Saw-Hee, F. L.,
Beevers, D. G. and Lip, G. Y. H. (2001) Plasma levels of
vascular endothelial growth factor and its soluble receptor
(SFlt-1) in essential hypertension. Am. J. Cardiol. 87,
805–807
10 Le Noble, F. A C., Staessen, F. R. M., Hacking, W. J. G. and
Boudier, A. J. S. (1998) Angiogenesis and hypertension.
J. Hypertens. 16, 1563–1572
11 Galen, F. X. (2002) Cell adhesion molecules in
hypertension: endothelial markers of vascular injury and
predictors of target organ damage? J. Hypertens. 20,
813–816
12 De Souza, C. A., Dengel, D. R., Macko, R. F., Cox, K. and
Seals, D. R. (1997) Elevated levels of circulating cell
adhesion molecules in uncomplicated essential
hypertension. Am. J. Hypertens. 10, 1335–1341
13 Yu, T., Mitchell, P., Berry, G., Li, W. and Wang, J. J. (1998)
Retinopathy in older persons without diabetes and its
relationship to hypertension. Arch. Ophthalmol. 116,
83–89
14 Wang, J. J., Mitchell, P., Leung, H., Rochtchina, E., Wong,
T. Y. and Klein, R. (2003) Hypertensive retinal vessel wall
signs in a general older population: the Blue Mountains
Eye Study. Hypertension 42, 534–541
15 Keith, N. M., Wagener, H. P. and Barker, N. W. (1939)
Some different types of essential hypertension: their course
and prognosis. Am. J. Med. Sci. 191, 332–343
16 Li, Y. H., Teng, J. K., Tsai, W. C., Tsai, L. M., Lin, L. J. and
Chen, J. H. (1997) Elevation of soluble adhesion molecules
is associated with the severity of myocardial damage in
acute myocardial infarction. Am. J. Cardiol. 80,
1218–1221
17 Tsai, W. C., Li, Y. H., Lin, C. C., Chao, T. H. and Chen,
J. H. (2004) Effects of oxidative stress on endothelial
function after a high-fat meal. Clin. Sci. 106, 315–319
18 Fleisch, M., Billinger, M., Eberli, F. R., Garachemani, A. R.,
Meier, B. and Seiler, C. (1999) Physiologically assessed
coronary collateral flow and intracoronary growth factor
concentrations in patients with 1- to 3-vessel coronary
artery disease. Circulation 100, 1945–1950

C 2005 The Biochemical Society