Download Influence of the 4G/5G PAI-1 genotype on angiotensin II

Cardiovascular Research 63 (2004) 176 – 185
www.elsevier.com/locate/cardiores
Influence of the 4G/5G PAI-1 genotype on angiotensin
II-stimulated human endothelial cells and in
patients with hypertension
C. Roncal a,b, J. Orbe a,b, J.A. Rodriguez a,b, M. Belzunce a,b, O. Beloqui c,
J. Diez b, J.A. Páramo a,b,*
a
Atherosclerosis Research Laboratory, School of Medicine, University of Navarra, 31080-Pamplona, Spain
b
Division of Cardiovascular Pathophysiology, Foundation for Applied Medical Research (FIMA),
School of Medicine, University of Navarra, Pamplona, Spain
c
University Hospital, University of Navarra, Pamplona, Spain
Received 1 December 2003; received in revised form 11 March 2004; accepted 18 March 2004
Available online 14 May 2004
Time for primary review 28 days
Abstract
Background: We examined the influence of the 4G/5G PAI-1 (plasminogen activator inhibitor) genotype on Angiotensin II (Ang II)induced PAI-1 expression by human endothelial cells (HUVEC) in the presence and absence of AT1-receptor blocker losartan, and screened
for this polymorphism in relation to plasma PAI-1 and arterial pressure in apparently healthy subjects. Methods and results: Genotyped
cultured HUVEC were incubated with Ang II (10 8 M) with or without losartan up to 24 h. PAI-1 mRNA was determined in cell extracts and
protein and activity assessed in supernatants and extracellular matrix (ECM). Ang II increased PAI-1 mRNA and activity in a genotypedependent manner, higher values observed for 4G/4G HUVEC compared with 4G/5G and 5G/5G genotypes ( p < 0.05). Laser confocal
microscopy and Western blot analysis showed increased PAI-1 protein within ECM in Ang II-stimulated cultures. PAI-1 expression and
protein secretion induced by Ang II in 4G/4G HUVEC was completely inhibited by preincubation with 0.05 AM losartan ( p < 0.01),
indicating an AT1-mediated effect. In a group of hypertensives homozygous for the 4G allele, PAI-1 antigen was significantly increased
(51.0 F 10.1 ng/ml) compared with normotensives (28.3 F 4.0 ng/ml) and hypertensives carrying the 5G allele ( p < 0.05). Conclusions: The
4G/5G PAI-1 polymorphism determines the endothelial PAI-1 upregulation by Ang II and the inhibitory response to losartan. Analysis of
PAI-1 genotypes may help identifying subgroups of hypertensives at higher cardiovascular risk.
D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: PAI-1; Atherosclerosis; Polymorphism; Endothelium; Angiotensin-II; Arterial hypertension
1. Introduction
Activation of the renin – angiotensin– aldosterone system
(RAAS) and defective fibrinolysis has been linked with an
increased thrombotic risk and fibrosis [1,2]. There is strong
evidence that the fibrinolytic balance is largely under the
control of the RAAS [3]. Angiotensin II (Ang II), a key
component of this system, plays a pivotal role in the
* Corresponding author. Atherosclerosis Research Laboratory, School
of Medicine, University of Navarra, 31080-Pamplona, Spain. Tel.: +34948296397; fax: +34-948296500.
E-mail address: [email protected] (J.A. Páramo).
homeostasis of systemic arterial pressure, and also modulates fibrinolysis by increasing type 1 plasminogen activator
inhibitor (PAI-1) in vitro and in vivo [4 –6]. PAI-1 is the
primary inhibitor of tissue-type plasminogen activator (tPA) and regulates fibrinolytic system [7]. A crucial role of
PAI-1 in atherothrombosis has been demonstrated both at
experimental and clinical levels [8– 10].
Endothelial cells in general, and human umbilical vein
endothelial cells (HUVEC) in particular, express PAI-1
[11,12]. Transcriptional control of PAI-1 promoter in response to cytokine and hormone stimuli is an important
regulatory mechanism to fibrinolysis. The PAI-1 4G/5G
polymorphism located 675 bp upstream of the start of
transcription has been associated with increased plasma
0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2004.03.023
C. Roncal et al. / Cardiovascular Research 63 (2004) 176–185
PAI-1 concentrations and with increased risk for coronary
disease [13 – 15]. It has been shown that proatherogenic
stimuli such as IL-1 and VLDL induced PAI-1 synthesis in
cells containing the 4G sequence [13,16], this effect being
explained through the binding of a transcriptional activator to
both 4G and 5G alleles, while the 5G allele also binds a
transcriptional repressor [13].
Whereas Ang II causes the release of PAI-1 by endothelial cells [5,6], no studies have addressed the issue of
whether those effects differ according to the 4G/5G endothelial cell genotype. Recent reports have also demonstrated
that the relationship between Ang II and PAI-1 would be
mediated by the angiotensin II type 1 (AT1) receptor
[17,18]; furthermore, AT1 receptor blockers have been
shown to attenuate angiotensin-induced inhibition of fibrinolysis and this may be one of the mechanisms in the
prevention of ischemic events [19]. In addition, AT1 receptor blockade has been shown to slow the progression of
vascular sclerosis via its effect on PAI-1 [20].
Because endothelial PAI-1 is an important source for PAI1 levels in plasma and there is evidence that genetic factors
may be important in determining inhibitor levels, we investigated whether the PAI-1 response by endothelial cells
challenged with Ang II is related to the 4G/5G genotype,
and whether selective AT1 receptor blockade would lower
endothelial cell production of PAI-1 in a genotype-specific
manner. In addition, the 4G/5G polymorphism and plasma
PAI-1 levels were also determined in apparently healthy
subjects in relation to their systemic arterial pressure.
2. Materials and methods
2.1. Endothelial cell culture
Endothelial cells (HUVEC) were isolated from human
umbilical cords obtained less than 8 h after delivery, by
digestion with collagenase A (Gibco) as previously described [11].
2.2. Experimental design
All studies were performed with confluent cultures in the
second passage. Cultures were washed with HBSS (Gibco)
after which fresh serum-free media (Gibco) supplemented
with penicillin/streptomycin was added. Cultures were challenged with Angiotensin II (Ang II, Sigma) 10 8 M alone,
or in the presence of AT1 receptor blocker losartan (0.01 and
0.05 AM, kindly provided by Merck, USA), preincubated 18
h, and supernatants and cells were harvested at 0, 2, 6 and
24 h after the start of the experiment. Dose of Ang II was
chosen according to previous dose –response assays (10 6
to 10 10 M, data not shown). Unstimulated cultures were
used as controls. Endothelial cells derived from three
individuals per genotype were used for each experimental
condition.
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2.3. Subjects
The population studied consisted of 122 consecutive
subjects attending the Vascular Risk Area of a single institution (University Clinic of Navarra) for global vascular risk
assessment. Blood pressure was measured twice on the right
upper arm with a random-zero mercury sphygmomanometer
in patients in the sitting position (average of two measurements). Forty six untreated subjects had elevated systolic
and/or diastolic blood pressure (>139/89 mm Hg) (hypertensive group) and 76 age-matched normotensive subjects
were used as control group. Body mass index (BMI) was
calculated as weight/height2 (kg/m2) and used as an estimate
of overall adiposity.
Written informed consent was obtained before participation in the study, and the local committee on human
research approved the study protocol. The study was performed in accordance with the principles of the Declaration
of Helsinki.
2.4. Laboratory analysis
Blood samples were collected on ice and centrifuged
immediately at 4 jC for 20 min. All plasma or serum was
separated and stored at 80 jC until assayed.
Serum C-reactive protein was assayed by hs-CRP immunoassay system (Immulyte hs-CRP, Diagnostic Product).
Serum aldosterone was measured using a commercially
available radioimmunoassay kit (Aldoctk-2, DiaSorin). The
intra- and inter-assay variation coefficients were 4% and
6%, respectively.
Serum cholesterol, HDL-cholesterol, LDL-cholesterol,
triglycerides and glucose were measured on fasting blood
samples by standard laboratory techniques.
2.5. PAI-1 promoter 4G/5G genotype
Genomic DNA was extracted from cord tissue samples or
subject blood samples using the TriPure Isolation Reagent
(Roche) following the manufacturer’s instructions. The PAI1 promoter 4G/5G polymorphism was analyzed with an
allele-specific PCR [21].
2.6. RNA isolation and real-time quantitative RT-PCR
Total RNA was extracted from cells with TriPure (Roche)
and quantified with ultraviolet spectroscopy. Total RNA (0.5
Ag) was reverse transcribed with random primers with MMLV reverse transcriptase (Invitrogen), in the presence of
RNase Outk (Invitrogen).
One hundred nanograms of reverse-transcribed RNA was
primed with specific oligonucleotides for PAI-1 (5VCCGTCTGATTTGTGGAAGAGG-3V and 5V-ACAGGAGGAGAAACCCAGCAG-3V), and h-actin (5V-AGCCTCGCCTTTGCCGA-3V and 5V-CTGGTGCCTGGGGCG-3V).
PCR was performed as previously described [22] using
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C. Roncal et al. / Cardiovascular Research 63 (2004) 176–185
SYBR Green PCR Master Mix (Applied Biosystems) on the
ABI PRISM 7000 Detection System (Applied Biosystems).
Potential genomic DNA contamination was excluded by
using intron-encompassing primers. All samples were
assayed in triplicate and normalized on the basis of their
h-actin content. A melting curve analysis and length verification by gel electroforesis were carried out to confirm the
specificity of PCR products.
2.10. Statistical analysis
Results are expressed as mean F S.E.M. of three independent experiments. For statistical purposes, all data at 2, 6 and
24 h were expressed as percentages of their baseline value,
which was considered 100%. Statistical analysis was performed by ANOVA or Kruskal – Wallis followed by Tukey-b
post-hoc test or Mann – Whitney U-test for comparisons
between genotypes. Differences between stimulated and
2.7. Determination of PAI-1 activity and antigen
Active PAI-1 was measured in HUVEC supernatants by
an amidolytic assay (Coatest-PAI, Chromogenix) as previously described [23]. PAI-1 antigen levels in culture supernatants and plasma samples were measured, by ELISA
(Asserachrom-PAI-1, Diagnostica Stago) according to manufacturer instructions.
2.8. Laser confocal immunofluorescence microscopy of
HUVEC
Cells grown on slides (Nalgene Nunc International) precoated with gelatin 0.1% were fixed with 4% paraformaldehyde and permeabilized with cold methanol/acetone (v/v)
before immunocytochemistry. Briefly, after incubating
HUVEC with monoclonal anti-PAI-1 (American Diagnostica, 20 Ag/ml) overnight at 4 jC, the slides were washed and
incubated for 1 h at room temperature with 10 Ag/ml Alexa
Fluor 488R anti-mouse IgG (Molecular Probes). TO-PROR3 (1.25 AM; Molecular Probes) was used for nuclear counterstaining. Finally, cells were mounted in SlowFadeR Antifade
reagent (Molecular Probes) and visualized under confocal
laser scan microscope (Zeiss LSM-510 Meta; 63 objective). Forty image stacks (512 512 pixels each) were
captured from representative areas. The specificity of the
fluorescent signal was checked in samples prepared by
omitting the first antibody.
2.9. PAI-1 protein in ECM
Genotyped HUVEC monolayers (n = 3) were washed
with cold PBS and incubated at 37 jC with 0.5% Triton
X-100 in PBS for 10 min. The supernatant was discarded and
the remaining ECM was solubilized with electrophoresis
(PAGE) sample buffer as previously described [24].
Samples from ECM were separated by SDS-PAGE under
reducing conditions in Tris – glycine –SDS buffer (pH 8.3).
Electrotransfer was performed in Tris – glycine – methanol
buffer for 1 h at 300 mA. Nitrocellulose membrane was
blocked for 1 h, then incubated with murine anti-PAI-1 (1 Ag/
ml, American Diagnostica) or rabbit anti-vitronectin (8 Ag/
ml, American Diagnostica) in PBS – Tween (0.1%). The
membrane was rinsed and incubated with a secondary antibody (anti IgG labeled with peroxidase, Amersham) and
developed by chemiluminiscence system (ECL, Amersham)
on X-ray film.
Fig. 1. Effect of Ang II on PAI-1 mRNA by cultured HUVEC (n = 3)
genotyped for the 4G/5G PAI-1 polymorphism. The time course of Ang IIinduced PAI-1 mRNA expression determined by real-time PCR in 4G/4G
(panel A), 4G/5G (panel B) and 5G/5G (panel C) cultures, is shown. Values
were normalized with h-actin and expressed as percentage of baseline.
y
p < 0.05 compared to basal; *p < 0.05 compared to unstimulated cultures.
C. Roncal et al. / Cardiovascular Research 63 (2004) 176–185
179
3. Results
3.1. Influence of the 4G/5G PAI-1 genotype on Ang II
induced PAI-1 expression by HUVEC
PAI-1 genotyped cultured HUVEC were stimulated
with 10 8 M Ang II, and the PAI-1 mRNA, as well
as activity and protein were measured before and 2, 6,
and 24 h after stimulation. The basal levels of PAI-1
mRNA (4G/4G: 36.6 F 0.6 AU, 4G/5G: 36.7 F 0.7 AU
Fig. 2. Effect of Ang II on PAI-1 activity by cultured HUVEC (n = 3)
genotyped for the 4G/5G PAI-1 promoter polymorphism. The time course of
Ang II-induced PAI-1 amidolytic activity in 4G/4G (panel A), 4G/5G (panel
B) and 5G/5G (panel C) endothelial cultures, is shown. Values are expressed
as percentage of baseline (mean F S.E.). yp < 0.05 compared to basal;
*p < 0.05 compared to unstimulated cultures.
unstimulated cultures of the same genotype were assessed by
Mann – Whitney U-test. Wilcoxon test was used to find
differences of paired data. Pearson or Spearman tests were
used for univariate associations of continuous variables.
Multiple regression analysis was performed to determine
whether PAI-1 genotype was an independent predictor of
plasma PAI-1 antigen. The statistical analysis was performed
with SPSS for Windows software package version 11.0.
Fig. 3. Effect of Ang II on PAI-1 antigen secretion by cultured HUVEC
(n = 3) genotyped for the 4G/5G PAI-1 polymorphism. The time course of
Ang II-induced PAI-1 antigen in 4G/4G (panel A), 4G/5G (panel B) and
5G/5G (panel C) cultures, is shown. Values are expressed as percentage of
baseline (mean F S.E.). yp < 0.05 compared to basal.
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C. Roncal et al. / Cardiovascular Research 63 (2004) 176–185
Fig. 4. Confocal microscope images of immunofluorescence staining for PAI-1 protein (pseudocolored in green) in 4G/4G HUVEC treated for 24 h with Ang II
(A), Ang II plus losartan (B) and unstimulated (C) cultures. A Z-section of each capture is shown at the top and the right hand side. No green fluorescence
signal was detected in the absence of the primary antibody (D). Nuclei were counterstained with TOPRO-3 (red).
and 5G/5G: 36.4 F 0.9 AU), PAI-1 activity (4G/4G:
30.3 F 4.6 U/ml, 4G/5G: 27.2 F 5.2 U/ml and 5G/5G:
2 1 . 4 F 3 . 8 U / m l ) a n d PA I - 1 a n t i g e n ( 4 G / 4 G :
1780.3 F 150.1 ng/ml, 4G/5G: 1553.5 F 167.0 ng/ml
and 5G/5G: 1668.7 F 130.6 ng/ml), did not differ among
genotypes.
Fig. 5. PAI-1 antigen was measured in ECM by Western blot analysis in unstimulated HUVEC (lanes 3 – 5), after Ang II stimulation (lanes 6 – 8), and in cells
pretreated with losartan 0.01 and 0.05 AM (lanes 9 – 14). Lane 1: molecular weight marker; lane 2: recombinant PAI-1. Panel B: Densitometric analysis of free
PAI-1 corresponding to three independent experiments. Free PAI-1 plus PAI-1/vitronectin were completely blocked by losartan 0.05 AM after 24 h Ang II
stimulation. Values are expressed as percentage of baseline (mean F S.E.). *p < 0.05 compared to unstimulated samples; zp < 0.05 compared to Ang IIstimulated samples.
C. Roncal et al. / Cardiovascular Research 63 (2004) 176–185
181
As shown in Fig. 1, Ang II induced a significant
increase of PAI-1 expression in 4G/4G HUVEC compared
to either baseline or unstimulated cultures. PAI-mRNA was
significantly elevated 2 h after stimulation (1.8-fold increase), the effect being maximal ( p < 0.05) at 6 h (2-fold
increase), to return to basal values after 24 h. However, the
addition of Ang II to 4G/5G and 5G/5G HUVEC was
followed by a slight non-significant increase in PAI-1
mRNA. As shown in Fig. 2A, changes in 4G/4G mRNA
paralleled those in PAI-1 activity (2.5-fold increase,
p < 0.05) at 2 (67.8 F 6.2 U/ml) and 6 h (59.8 F 5.1 U/
ml) compared to baseline (24.3 F 3.3 U/ml), activity levels
still remaining elevated (3-fold increase) at 24 h (72.8 F 5.7
U/ml). The addition of Ang II to 4G/5G HUVEC was
followed by a lower increase in PAI-1 activity at 2 and 6
h ( p = 0.07), without differences in the activity for the 5G/
5G cultures. Finally, no significant increase in the amount
of PAI-1 released into medium was observed (Fig. 3) for
all tested endothelial genotypes as compared with unstimulated cultures, further confirmed by Western blot analysis of supernatants (data not shown).
The influence of genotype on the magnitude of response
to Ang II was investigated by studying the ratio of Ang II
stimulated/basal levels in the different HUVEC genotyped
cultures. Mean PAI-1 mRNA and activity were respectively
1.5- and 1.9-fold higher in 4G/4G cultures compared to the
remaining genotypes.
Since no significant increase in the PAI-1 protein
secreted into the medium could be detected after Ang II
stimulation, further experiments were carried out to assess
the amount of PAI-1 present in the cells and ECM. As
shown in Fig. 4, laser confocal microscopy revealed a
significant increase of PAI-1 protein in the basal surface
of the cells after Ang II stimulation. Western blot analysis
of ECM samples showed a significant increase of free
PAI-1 (50 kDa) and PAI-1/vitronectin complexes (112
kDa) in 4G/4G HUVEC, 6 and 24 h after Ang II
stimulation (Fig. 5).
3.2. Effect of losartan on Ang II-induced PAI-1 response by
4G/4G HUVEC cultures
In a further step, the involvement of AT1 receptor on
Ang II-induced PAI-1 expression in 4G/4G genotyped
cultures was assessed by preincubation (18 h) with different
losartan concentrations (0.01 – 0.05 AM). As shown in Fig.
6, the Ang II-induced PAI-1 mRNA was completely neutralized by the higher losartan dose (0.05 AM) after 6-h Ang
II addition (220.9 F 10.7% vs. 116.2 F 20.7%, p < 0.05).
This dose of losartan also reduced significantly the PAI-1
activity into the medium throughout the experiment. Moreover, a marked reduction in PAI-1 protein in the basal
surface of cells (Fig. 4) and in the amount of free plus
complexed PAI-1 within ECM was found in losartantreated HUVEC 6 h after Ang II stimulation, with complete
inhibition after 24 h (Fig. 5).
Fig. 6. Effect of losartan on Ang II-induced PAI-1 expression by 4G/4G
genotyped cultures (n = 3). The Ang II-induced PAI-1 mRNA (A), PAI-1
activity (B) and antigen (C) in HUVEC pretreated with losartan (0.01 – 0.05
AM) is shown. Values are expressed as percentage compared to baseline
(mean F S.E.). The high losartan dose (0.05 AM) completely blocked the
Ang II-induced PAI-1 mRNA at 6 h and activity at 6 and 24 h. yp < 0.05
compared to basal; zp < 0.05 compared to Ang II.
3.3. 4G/5G PAI-1 polymorphism and protein levels in
hypertensive subjects
The possible interactions among the 4G/5G PAI-1 polymorphism, plasma PAI-1 antigen levels and arterial blood
pressure was assessed in 122 apparently healthy subjects
(mean age 50.5 F 0.8 years, 84% men) referred to our
182
C. Roncal et al. / Cardiovascular Research 63 (2004) 176–185
Table 1
Partial correlation between PAI-1 levels and the clinical and biochemical
parameters analyzed after controlling for age and sex
BMI (kg/m2)
SBP (mm Hg)
DBP (mm Hg)
Total cholesterol (mg/dl)
HDL-cholesterol (mg/dl)
LDL-cholesterol (mg/dl)
Triglycerides (mg/dl)a
CRP (mg/l)a
Glucose levels (mg/dl)
Aldosterone (pg/ml)
R
P
0.25
0.07
0.10
0.11
0.18
0.13
0.20
0.19
0.31
0.25
0.01
0.38
0.25
0.20
0.04
0.15
0.02
0.02
0.01
0.01
BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood
pressure; CRP: C-reactive protein.
a
Logaritmically transformed variables to normalized their distribution.
28.6%), consistent with the Hardy –Weinberg equilibrium,
and did not differ from other series [25]. The 4G/5G PAI-1
genotypes percentage and the presence of other cardiovascular risk factors were similar between groups (Table 2).
Moreover, the 4G/4G hypertensive group had significantly
higher plasma PAI-1 (51.0 F 10.1 ng/ml) than either 4G/4G
normotensives (28.3 F 4.0 ng/ml) or hypertensives with the
5G allele ( p < 0.05). Multiple linear regression analysis
showed that the PAI-1 genotype (4G/4G vs. 4G/5G plus
5G/5G) was a significant predictor of plasma PAI-1 antigen
in hypertensives after adjustment for age, sex, glucose
levels, BMI, CRP, dyslipidemia and smoking habits
(r = 0.46, p < 0.01).
4. Discussion
institution for global vascular risk assessment. In the overall
population, hypertensive patients (n = 46) had significantly
higher BMI (29.2 F 4.2 vs. 26.2 F 3.6, p < 0.01) and PAI-1
antigen (39.0 F 3.8 vs. 30.7 F 2.4 ng/ml, p < 0.05) than
normotensive subjects (n = 76). Moreover, serum aldosterone
levels were also significantly increased in hypertensives
(176.9 F 78.1 vs. 150.0 F 59.0 pg/ml, p < 0.03). As shown
in Table 1, there was a significant positive correlation
between plasma PAI-1 levels, several biochemical parameters and markers of atherosclerosis. After controlling for age
and sex, the associations remained for BMI ( p < 0.01),
glucose ( p < 0.01), HDL-cholesterol ( p < 0.04), triglycerides
( p < 0.02), CRP ( p < 0.02) and serum aldosterone ( p < 0.01).
PAI-1 genotype frequencies were: 4G/4G (n = 36,
29.5%), 4G/5G (n = 51, 41.8%) and 5G/5G (n = 35,
This study demonstrates a genotype-specific regulation
of PAI-1 in Ang II-stimulated HUVEC, the 4G/4G PAI-1
genotype being critical in endothelial PAI-1 response
through the AT1 receptor, since the effect was completely
prevented by losartan. In addition, higher PAI-1 antigen
levels were found in hypertensive subjects homozygous for
the 4G allele compared to the remaining genotypes. Taken
together, our data strongly support a significant role for the
4G/5G PAI-1 genotype in determining PAI-1 response in
arterial hypertension.
The proatherogenic effects of Ang II appear to be
partially related to reduced fibrinolysis [3,26]. PAI-1 is
considered to be an important regulator of fibrinolysis and
ECM turnover [7]. In addition, PAI-1 plays a crucial role in
the development of atherosclerosis and neointimal forma-
Table 2
Clinical characteristics of the studied population according to 4G/5G PAI-1 polymorphism (mean F S.E.M. is shown)
4G/4G
Age, years
Sex (M/F)
BMI, kg/m2
Smokers (%)
Diabetes (%)
Systolic blood
pressure, mm Hg
Diastolic blood
pressure, mm Hg
Glucose, mg/dl
Total cholesterol,
mg/dl
HDL cholesterol,
mg/dl
Triglycerides, mg/dl
CRP, mg/l
PAI-1 antigen, ng/ml
4G/5G
5G/5G
Normotensives
(n = 24)
Hypertensives
(n = 12)
Normotensives
(n = 27)
Hypertensives
(n = 24)
Normotensives
(n = 25)
Hypertensives
(n = 10)
47.7 F 2.9
22/2
26.2 F 0.9
50
5
117.5 F 2.5
53.3 F 1.7
11/1
29.3 F 1.1*
42
16
138.8 F 2.9**
48.6 F 1.8
22/5
26.8 F 0.6
48
4
115.6 F 1.8
52.7 F 1.7
20/4
28.7 F 0.9*
42
12
150.0 F 3.3**
48.0 F 1.9
19/6
24.7 F 0.6
40
2
113.0 F 2.2
54.0 F 2.9
7/3
29.1 F 1.1**
40
8
148.0 F 3.5**
76.1 F 1.5
88.7 F 2.1**
76.1 F 1.3
92.5 F 1.5**
76.5 F 1.3
91.5 F 3.6**
98.6 F 2.7
235.4 F 8.7
108.7 F 11.5
240.1 F 7.7
103.6 F 5.4
240.0 F 6.3
106.4 F 6.2
258.2 F 8.8
94.8 F 2.1
233.4 F 8.9
95.3 F 1.9
246.8 F 13.3
54.9 F 2.8
52.2 F 3.8
50.9 F 2.2
47.4 F 2.3
58.1 F 3.4
55.9 F 2.1
109.0 F 10.8
3.1 F 2.5
28.3 F 4.0
107.2 F 8.9
3.1 F 1.7
51.0 F 10.1*
118.4 F 15.4
2.4 F 1.6
30.9 F 4.3
141.8 F 16.4
4.4 F 4.1
29.6 F 2.9y
111.5 F 11.6
2.5 F 3.2
31.5 F 5.1
147.5 F 22.9
3.4 F 2.6
26.6 F 5.1y
* p < 0.05 compared to normotensive.
** p < 0.01 compared to normotensive.
y
p < 0.05 compared to 4G/4G hypertensives.
C. Roncal et al. / Cardiovascular Research 63 (2004) 176–185
tion after vascular injury [27,28]. Because of the association
of PAI-1 gene 4G/5G polymorphism with circulating PAI-1
levels [14,15], we first tested the hypothesis that Ang IIinduced PAI-1 by HUVEC would be dependent on the
endothelial 4G/5G genotype. Ang II significantly increased
the PAI-1 gene expression in cultures with 4G/4G genotype
compared to 5G allele.
The exact mechanism by which Ang II might regulate
endothelial PAI-1 expression in a genotype-specific manner
has not been clearly defined. It can be speculated that the
4G/4G genotype, in the absence of repressor binding site,
would elicit increased response to Ang II through nuclear
transcription factor kB (NF-kB) pathway [13]. Ang II
response sites have been identified upstream the PAI-1
promoter [29] involving signaling cascades depending on
PKC activation, which in turn can activate NF-kB in
endothelial cells [30]. The competition between the transcriptional factor and the 5G allele-specific transcriptional
repressor protein might explain the increased PAI-1 response by HUVEC 4G/4G upon Ang II stimulation. Additional studies are, however, needed to examine how this
factor might influence Ang II-induced PAI-1 at the promoter
level. Previous studies have associated the homozygosity for
the 4G allele with PAI-1 after RAAS activation by salt
depletion [31], with triglycerides [14] and with circadian
variations in PAI-1 [15].
The PAI-1 mRNA expression paralleled an increase in
PAI-1 activity, but not in the amount of protein released into
the medium suggesting post-translational modifications.
Previous studies reporting elevated PAI-1 antigen after
Ang II stimulation could be related to different endothelial
origin [32] or experimental conditions. In fact, a slight but
significant increase in Ang II-stimulated HUVEC was only
observed with 100 times higher Ang II concentrations [33].
However, our data would agree with a previous study
showing that calcium mobilizing agents (as Ang II) may
indeed suppress the proinflammatory cytokines-induced
increase in PAI-1 synthesis due to impaired protein translation [34].
Quite unexpectedly, we found that the majority of Ang
II-induced PAI-1 antigen was localized within ECM. The
relationship between Ang II and cardiovascular remodeling
has not been clearly defined. Several studies have demonstrated that Ang II can modulate synthesis and degradation
of ECM components [35,36]. Our results indicate that it
may also favor the endothelial secretion of PAI-1 towards
the subendothelial space, as demonstrated by the increased
presence of free PAI-1 and PAI-1/vitronectin (Vn) complexes in ECM. PAI-1 and Vn are known to participate in
the early stages of the vascular response to injury by
stabilizing the initial thrombus and preventing early fibrinolysis [37] but are also involved in pathological fibrosis
[38]. The presence of free and Vn-complexed PAI-1 within
ECM in Ang II-stimulated HUVEC might reflect the limitation of excessive proteolysis favoring fibrous tissue deposition [39].
183
We found that Ang II-induced PAI-1 by HUVEC in
4G/4G could be prevented by losartan. Whereas it has
been previously reported that the 4G/5G genotype influenced the change in PAI-1 during ACE inhibition [40], no
studies have demonstrated a similar effect of AT1 receptor
blockers. Interestingly, a dose of losartan lower than
required to reduce blood pressure in humans (0.05 AM)
completely blocked Ang II-induced PAI-1 expression and
this may be one of the mechanisms by which losartan
may decrease the thrombotic potential of the vascular wall
and increase fibrinolysis [41]. Controversy still exists,
however, as to the effects of short- and long-term interruption of the RAAS system by either ACE inhibition or
AT1 receptor antagonists on plasma fibrinolytic balance
[41 –44].
The reduced amount of PAI-1 and PAI-1/Vn complexes
within ECM in losartan-treated 4G/4G HUVEC would
support a role for AT1 receptor blockade in the modulation
of vascular fibrotic processes, as suggested by in vivo
experiments [20].
In a next step, we investigated the relationship between
PAI-1 polymorphism and PAI-1 antigen in relation to
arterial hypertension, in subjects free of clinical cardiovascular disease. PAI-1 antigen was significantly increased in
4G/4G hypertensives compared to normotensives and
hypertensives carrying the 5G allele. Results remained
significant after adjusting for important confounding variables, such as BMI, triglycerides, glucose and CRP, thus
emphasizing the pathophysiological relation between PAI-1
polymorphism, PAI-1 antigen and arterial hypertension. The
observed correlation between the PAI-1 and aldosterone
agrees with a previous report indicating that aldosterone is
a major contributor of variability in PAI-1 levels in hypertensive subjects [26].
Impaired fibrinolysis due to elevated PAI-1 has been
reported as a marker of early atherothrombosis in hypertensives [45]. Moreover, it has been recently pointed out a
positive association between 4G/5G PAI-1 genotype and
plasma PAI-1 activity in Chinese patients with arterial
hypertension [46]. Some metabolic factors, such as insulin
resistance and obesity, may also contribute to the high level
of PAI-1 [47]. However, in the series analyzed, an independent association of PAI-1 and the 4G/5G polymorphism was
observed in hypertensives, after adjustment for BMI and
other metabolic factors.
In conclusion, this study shows that Ang II-induced
PAI-1 expression by HUVEC is dependent on the 4G/5G
cell genotype and can be effectively prevented by losartan in 4G/4G cultures. The common 675 4G/5G
polymorphism in the PAI-1 gene was associated with
PAI-1 levels in hypertension, since hypertensive carriers
of the 4G allele in its homozygous form had greater
circulating PAI-1 antigen levels. Reduced fibrinolytic
potential related to high PAI-1 might be an additional
cardiovascular risk factor in 4G/4G homozygous with
arterial hypertension.
184
C. Roncal et al. / Cardiovascular Research 63 (2004) 176–185
Acknowledgements
This project was funded through the agreement between
FIMA and the ‘‘UTE project CIMA’’.
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