Abstract

Background:

C-reactive protein (CRP) predicts cardiovascular outcome. Oxidative stress is considered to be involved in endothelial alteration. We hypothesized that in essential hypertension (EH), oxidative stress, as measured by 8-iso-prostaglandin-F (8-iso-PGF), should be associated with increased CRP and endothelial activation, as evaluated by soluble intercellular adhesion molecule–1 (ICAM-1) and vascular adhesion molecule–1 (VCAM-1) plasma levels.

Methods:

In 83 subjects with mild EH and in 50 healthy control subjects we measured, in basal conditions, plasma levels of hs-CRP, 8-iso-PGF, ICAM-1 and VCAM-1, and tumor necrosis factor–α (TNF-α).

Results:

Subjects with EH had higher levels of 8-iso-PGF (P < .0001), CRP (P < .001), ICAM-1 and VCAM-1 (P < .001), and TNF-α (P < .001) than did control subjects. We divided successively EH according to CRP values (<1, 1–3, >3 mg/L), and we observed increasing and significantly different levels of the endothelial parameters and of TNF-α along with increasing CRP. Linear analysis of correlation pointed out significant correlation of CRP with 8-iso-PGF (r = 0.730, P < .001), ICAM-1 and VCAM-1 (r = 0.642 and 0.468, P < .001 respectively), and TNF-α (r = 0.609, P < .001).

Results:

Multiple regression analysis using CRP as a dependent variable confirmed the relationship of CRP with systolic blood pressure (β 0.216, P = 0.039) and with 8-iso-PGF (β 0.602, P = .0001).

Conclusions:

Our data demonstrate that in EH, inflammatory molecules such as CRP and TNF-α are increased and related to both oxidative stress and endothelial activation.

Endothelial dysfunction is characterized by a shift of the actions of the endothelium toward reduced vasodilation, and both pro-thrombotic properties and pro-inflammatory state have been reported.1 Inflammation has also been associated with decreased endothelium-dependent relaxation, a process related to an alteration in the bioavailability of nitric oxide.1 There is considerable evidence that both endothelial dysfunction and inflammation are associated with most forms of cardiovascular disease such as essential hypertension and coronary artery disease as well as with chronic renal failure.2–5 Besides, there are experimental evidences that oxidative stress contributes to the pathogenesis of hypertension and may be involved in the process of atherogenesis.6

C-reactive protein (CRP), a sensitive marker of inflammation, is thought to represent a state of chronic low-grade inflammation of the arterial vessel wall at atherosclerotic sites. Consistently CRP predicts cardiovascular outcome.7–9

The CRP is present in the vessel wall, where it induces expression of the adhesion molecules vascular adhesion molecule–1 (VCAM-1) and intercellular adhesion molecule–1 (ICAM-1) by endothelial cells and serves as a chemoattractant for monocytes.10 The CRP binds to plasma membranes of damaged cells and activates complement via the classical pathway; and an intact complement system seems to be crucial for maturation of atherosclerotic lesions.11 The CRP is associated with endothelial cell dysfunction and progression of atherosclerosis,12 possibly by decreasing nitric oxide synthesis.13

In endothelial dysfunction, mechanisms that participate in the reduced vasodilatory responses include reduced nitric oxide generation and oxidative excess. Upregulation of adhesion molecules participates in the inflammatory response.

The isoprostane 8-iso-prostaglandin-F (8-iso-PGF), an index of lipid peroxidation endowed with vasoconstrictive and platelet-activating properties, is considered to be a surrogate for increased reactive oxygen species (ROS) production14; therefore it is considered a useful index of oxidative stress.15

We hypothesized that in EH inflammatory molecules such as CRP and TNF-α would be increased and associated with oxidative stress, as measured by 8-iso-PGF2α, and with endothelial activation as analyzed by soluble forms of ICAM-1 and VCAM-1.

Methods

In accordance with the Declaration of Helsinki and institutional guidelines, the protocol was approved by the local Ethical Committee. Subjects were aware of the investigational nature of the study and gave oral consent to participate.

Study Population

We considered 83 patients with never-treated essential hypertension entering the program. Patients were classified as hypertensive when clinic blood pressure (BP) was ≥140 mm Hg for systolic BP (SBP) and ≥90 mm Hg for diastolic BP (DBP); and severity of hypertension was defined according to the Seventh Report of the Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure.16

Clinic BP was considered as the average of three consecutive measurements using a mercury sphygmomanometer after the subject had been supine for 5 min.

Secondary or complicated forms of hypertension were ruled out by clinical examination, determination of serum creatinine, serum and urinary electrolytes, plasma catecholamine levels, renin activity, and renal echography.

Exclusion criteria were as follows: age <30 years or >65 years; diabetes mellitus type 1 or 2; previously diagnosed secondary or accelerated-malignant arterial hypertension; history of stroke, TIA, coronary heart disease, or myocardial infarction; abnormalities of cardiac rhythm or conduction under pharmacologic treatment; cardiac failure; and serum creatinine >1.3 mg/dL in women and >1.5 mg/dL in men. Patients taking nonsteroidal anti-inflammatory drugs discontinued the medications 4 weeks before entering into the study.

Study Protocol

On the day of the study, at 9 AM with the patients in supine position and after an overnight fast, blood samples were obtained from an in-dwelling forearm venous catheter to assay soluble forms of adhesion molecules ICAM-1 and VCAM-1, CRP, TNF-α, and 8-iso-PGF.

A total of 50 healthy subjects who were age matched to the patients were enrolled as control subjects.

Laboratory Methods

All endothelium-derived parameters were measured by solid-phase, specific sandwich enzyme-linked immunosorbent assay (ELISA). Standard curves were constructed using appropriated concentrations for each factor. Precautions were taken to avoid interference with other serum components.17

High-sensitivity CRP was measured by commercially available ELISA kit (Diagnostic Biochem, London, Ontario, Canada). The sensitivity was 10 ng/mL, the interassay coefficient of variation <10%, and the intra-assay coefficient of variation <8%.

Adhesion molecules assay (Bender MedSystem Diagnostics GmbH, Vienna, Austria) had sensitivities of 3.3 and 0.9 ng/mL for ICAM-1 and VCAM-1, respectively. The CV were 4% and 3.1%, respectively.

The TNF was assayed by Amersham Bioscences kit (Little Chalfont, England). The sensitivity was <5 pg/mL, and the reproducibility of both intra- and interassay had a coefficient variation <10%.

Oxidative stress measured as 8-iso-PGF was analyzed by commercial kit (Assay Design Inc., Ann Arbor, MI). Sensitivity was 16.3 pg/mL; the interassay coefficient of variation was <9%.

Statistical Analysis

After dividing the overall group into three subgroups according to CRP levels,9 differences between groups were evaluated using analysis of variance and the Student t test corrected with Bonferroni correction for multiple comparison. Simple and multiple regression analyses were used to test the relationships between adhesion molecules and other variables.

The multiple regression analysis was carried out considering CRP as dependent variable, in a model comprising body mass index, serum creatinine, glucose and cholesterol values, BP, adhesion molecules, TNF-α, and 8-iso-PGF. Results are given as means ± SD. The null hypothesis was rejected at a two-tailed P value of ≤ .05. The statistical analyses were performed using the SYSTAT DATA software package, version 5.2 (Systat, Evanston, IL).

Results

Table 1 gives demographic data of healthy control subjects and patients with EH. The comparison between control subjects and EH demonstrated that in hypertensive patients, plasma levels of both adhesion molecules CRP and TNF-α were increased, as was oxidative stress as measured as 8-iso-PGF (Table 1).

Table 1

Clinical data of healthy control subjects and essential hypertensive patients

 Control subjects (n = 50) Essential hypertensive patients (n = 83) 
Age (years) 45 ± 10 43.8 ± 11 
BMI 25.6 ± 2.8 27.8 ± 3.87 
SBP (mm Hg) 112 ± 4 146 ± 4.3* 
DBP (mm Hg) 73 ± 4 95.2 ± 4.2* 
s Cholesterol (mg/dL) 201 ± 12 193 ± 39 
s Trygliceride (mg/dL) 102 ± 14 117 ± 39 
HDL Cholesterol(mg/dL) 47 ± 3 44 ± 10.6 
s Glucose (mg/dL) 89 ± 10 94.7 ± 9 
S Creatinine (mg/dL) 0.91 ± 0.09 0.93 ± 0.15 
CRP (mg/L) 1.6 ± 0.4 2.37 ± 0.57* 
TNF-α (pg/mL) 1.6 ± 0.45 3.2 ± 0.4* 
sICAM-1 (ng/mL) 198.6 ± 30 288 ± 61* 
sVCAM-1 (ng/mL) 794 ± 89 951 ± 160* 
8-Iso-PGF (pg/mL) 86 ± 9.8 190 ± 63* 
 Control subjects (n = 50) Essential hypertensive patients (n = 83) 
Age (years) 45 ± 10 43.8 ± 11 
BMI 25.6 ± 2.8 27.8 ± 3.87 
SBP (mm Hg) 112 ± 4 146 ± 4.3* 
DBP (mm Hg) 73 ± 4 95.2 ± 4.2* 
s Cholesterol (mg/dL) 201 ± 12 193 ± 39 
s Trygliceride (mg/dL) 102 ± 14 117 ± 39 
HDL Cholesterol(mg/dL) 47 ± 3 44 ± 10.6 
s Glucose (mg/dL) 89 ± 10 94.7 ± 9 
S Creatinine (mg/dL) 0.91 ± 0.09 0.93 ± 0.15 
CRP (mg/L) 1.6 ± 0.4 2.37 ± 0.57* 
TNF-α (pg/mL) 1.6 ± 0.45 3.2 ± 0.4* 
sICAM-1 (ng/mL) 198.6 ± 30 288 ± 61* 
sVCAM-1 (ng/mL) 794 ± 89 951 ± 160* 
8-Iso-PGF (pg/mL) 86 ± 9.8 190 ± 63* 

BMI = body mass index; CRP = C-reactive protein; DBP = diastolic blood pressure; HDL = high-density lipoprotein; SBP = systolic blood pressure; sICAM = soluble intercellular adhesion molecule; sVCAM = soluble vascular adhesion molecule; TNF = tumor necrosis factor.

*

P < .001 controls v hypertensive subjects.

As shown in Figure 1, we divided successively the overall group according to CRP levels (<1, 1 to 3, and >3 mg/L), and we observed increasing levels of all endothelial parameters along with increasing CRP, with significant differences between groups in each endothelial parameter.

Mean plasma levels of intercellular adhesion molecule–1 (ICAM-1) and vascular adhesion molecule–1 (VCAM-1), tumor necrosis factor–α (TNF-α), and 8-iso-prostaglandin-F (8-iso-PGF) divided according to plasma levels of CRP in patients with essential hypertension.

Moreover, even in the lower CRP group the mean values of all examined molecules were significantly increased in comparison with data from healthy control subjects (8-iso-PGF ± 124.7 ± 23 v 86 ± 9.8 P < .01; ICAM-1: 245 ± 27 v 198.6 ± 30, P < .01; VCAM-1: 856.7 ± 72 v 794 ± 89 ng/mL, P < .01; TNF-α: 2.84 ± 0.35 v 1.6 ± 0.45, P < .01).

To verify these results we divided successively EH according to CRP tertiles, and we observed increasing levels of all parameters along with CRP tertiles, with significant differences among tertiles in each parameter (data not shown).

Univariate and Multivariate Analyses of Correlation

In EH the linear analysis of correlation pointed out significant correlations of CRP with 8-iso-PGF (r = 0.730, P < .001) (Fig. 2), ICAM-1 and VCAM-1 (r = 0.642 and 0.468, P < .001 respectively) (Fig. 3), and TNF-α (r = 0.609, P < .001). The latter in turn was significantly correlated with 8-iso-PGF (r = 0.757, P < .001), and with both ICAM-1 (r = 0.839, P < .001) and VCAM-1 (r = 0.701, P < .001).

Correlation between C-reactive protein (CRP) and soluble intracellular adhesion molecule–1 (ICAM-1) plasma levels in normal control subjects (A) and in the overall group of patients with essential hypertension (B).

Correlation between oxidative stress as measured as 8-iso-prostaglandin-F (8-iso-PGF) and C-reactive-protein (CRP) levels in the normal control subjects (A) and in the overall group of patients with essential hypertension (B).

The analysis of the relation of oxidative stress with atherogenic factors pointed out significant correlation of 8-iso-PGF with ICAM-1 and VCAM-1 (r = 0.828 and 0.596, P < .001, respectively).

In the control group, CRP correlated significantly with 8-iso-PGF, whereas no correlation with adhesion molecules and BP was found (Figs. 2 and 3).

In addition, SBP correlated with both 8-iso-PGF (r = 0.721, P < .001) and CRP (r = 0.611, P < .001) (Fig. 4) as well as DBP was correlated with 8-iso-PGF (r = 0.596, P < .001) and with CRP (r = 0.403, P < .001).

Correlation between C-reactive-protein (CRP) plasma levels and systolic blood pressure (SBP) in normal control subjects (A) and in the overall group of patients with essential hypertension (B).

Multiple regression analysis using CRP as a dependent variable, in a model that included BMI, serum creatinine, glucose and cholesterol values, BP, adhesion molecules, TNF-α, and 8-iso-PGF, showed a significant relationship of CRP with SBP (β 0.216, P = .039) and with 8-iso-PGF (β 0.602, P = .001).

Discussion

The present study shows in vivo that oxidative stress, as measured by 8-iso-PGF and atherogenic factors along with TNF-α increase in a continuum fashion with increasing CRP levels. Therefore, our data demonstrate the relationship between inflammation, oxidative stress, and atherogenic activation in human essential hypertension.

C-reactive protein is an acknowledged indicator of increased systemic inflammation, and inflammation is believed to be a key process in atheroma formation. The substance CRP has been reported to decrease production of nitric oxide by endothelial cells13 and to have properties of up-regulating angiotensin type–1 receptor expression18 affecting the renin-angiotensin system, and contributing to hypertensive disease. In addition, CRP has been shown to stimulate endothelin-1 and to induce adhesion molecule expression in human endothelial cells.19 These phenomena are all indicative of progressive endothelial inflammation and atherosclerosis.7

In our study CRP levels were significantly increased in EH when compared with healthy control subjects, and were significantly correlated to both systolic and diastolic BP. On the contrary, in normotensive control subjects CRP did not correlate with BP. These data seem to be in line with what reported previously regarding the relationship between CRP values, increasing categories of BP levels,9 and risk of developing arterial hypertension.20

In particular, in EH, the multiple regression analysis carried out considering CRP as a dependent variable disclosed a good correlation between CRP and systolic BP. This finding further highlights the linkage between BP levels and inflammation.

In EH associated with increased values of CRP we observed augmented levels of TNF-α, correlating with each other.

Tumor necrosis factor–α is essentially produced by monocytes and macrophages. In turn it is endowed with the capability of inducing endothelial activation, and it is the strongest known paracrine activator of monocytes and macrophages.15 Both TNF-α and CRP are found in considerable quantities in atherosclerotic lesions.21,22 Our findings seem to confirm the relation of these two inflammatory indices.

More data supporting the linkage of increased inflammatory markers with atherogenesis is given by our finding of augmented levels of soluble fraction of adhesion molecules, which correlated with both CRP and TNF-α. It was experimentally shown that CRP induces ICAM-1 and VCAM-1 expression in human endothelial cells in the presence of serum.19 The effect of CRP on both adhesion molecules was already present at a concentration of 5 μg/mL and was maximal at 50 μg/mL. The substance CRP is an acute-phase reactant usually present in human serum with a concentration <1 μg/mL. In patients with angina, serum levels >3 μg/mL are associated with increased risk of coronary events, but even small increases in serum levels of CRP are associated with higher risk of atherosclerosis.19 In line with this report, we observed that both ICAM-1 and VCAM-1 were significantly increased even in the group with lower CRP values.

Although there is now strong evidence that serum CRP levels are an independent risk factor for cardiovascular disease, the mechanisms underlying this association are uncertain.

In animal models of hypertension, oxidative excess leads to endothelial dysfunction as evidenced by improvement of the impaired endothelium-dependent relaxation after use of antioxidants.23 Oxidative excess in hypertensive patients leads to diminished nitric oxide24 and correlates with the degree of impairment of endothelium-dependent vasodilation and with cardiovascular events.25 Low bioavailability of nitric oxide can upregulate VCAM-1 in the endothelial cell layer via induction of nuclear factor–κB expression.26 In addition ROS, CRP, and CD40 ligand upregulate endothelial expression of adhesion molecules.10 The expression of VCAM-1 and ICAM-1 plays a role in the initiation of the inflammatory process.

The substance 8-iso-PGF, an index of lipid peroxidation endowed with vasoconstrictive and platelet-activating properties, is considered to be a surrogate for increased ROS production and an index of oxidative stress.14,15 Urinary 8-iso-PGF is increased in subjects at risk for future cardiovascular events.14

Some investigators27,28 have reported that hypertensive subjects are variably, but often not, undergoing increased oxidative stress. However Minuz et al29 found that patients with hypertensive disease had elevated excretion of both 8-iso-PGF and thromboxane, suggesting that enhanced oxidative stress and platelet activation are increased in this population.

Our results are in line with those of Minuz et al.29 Oxidative stress was evaluated by 8-iso-PGF assay, demonstrating increased serum levels of this index, which correlated with both inflammatory and endothelial activation markers. Besides, in the statistical model that included BMI, BP. 8-iso-PGF, TNF-α, adhesion molecules and serum creatinine, glucose and cholesterol, and included CRP as the dependent variable, 8-iso-PGF was closely related to CRP. Therefore, it is conceivable that oxidative stress, either resulting from or acting together with SBP, contributes in triggering the inflammatory process in essential hypertension.

A limitation of the present study is the assessment of circulating molecules, considered as promising tools for evaluating cardiovascular risk,15,30 even if their levels could be affected by several clinical conditions and drugs. Therefore, potential bias must be considered. Furthermore, with regard to isoprostanes quantification, several methods have been developed, the most sensitive and specific of which is mass spectrometry. Nonetheless, this method requires considerable expenditure in equipment, and is labor intensive.

In conclusion, our data demonstrate that in essential hypertension inflammatory molecules such as CRP and TNF-α are increased and are related to both oxidative stress and endothelial activation. These observations suggest that in established essential hypertension a better knowledge of the mechanisms involved in oxidative stress could be a key to understanding the links among inflammation, endothelial activation and atherosclerosis.

References

1.
Kharbanda
RK
,
Walton
B
,
Allen
M
,
Klein
N
,
Hingorani
AD
,
MacAllister
RJ
,
Vallance
P
:
Prevention of inflammation-induced endothelial dysfunction
.
Circulation
 
2002
;
105
:
2600
2604
.
2.
Park
JB
,
Charbonneau
F
,
Schiffrin
EL
:
Correlation of endothelial function in large and small arteries in human essential hypertension
.
J Hypertens
 
2001
;
19
:
415
420
.
3.
Monnink
SH
,
van Haelst
PL
,
van Boven
AJ
,
Stroes
ES
,
Tio
RA
,
Plokker
TW
,
Smit
AJ
,
Veeger
NJ
,
Crijns
HJ
,
van Gilst
WH
:
Endothelial dysfunction in patients with coronary artery disease: a comparison of three frequently reported tests
.
J Invest Med
 
2002
;
50
:
19
24
.
4.
Bolton
CH
,
Downs
LG
,
Victory
JG
,
Dwight
JF
,
Tomson
CR
,
Mackness
MI
,
Pinkney
JH
:
Endothelial dysfunction in chronic renal failure: roles of lipoprotein oxidation and pro-inflammatory cytokines
.
Nephrol Dial Transplant
 
2001
;
16
:
1189
1197
.
5.
Cottone
S
,
Mulè
G
,
Amato
F
,
Riccobene
R
,
Vadalà
A
,
Lorito
MC
,
Raspanti
F
,
Cerasola
G
:
Amplified biochemical activation of endothelial function in hypertension associated with moderate to severe renal failure
.
J Nephrol
 
2002
;
15
:
643
648
.
6.
Kunsch
C
,
Medford
RM
:
Oxidative stress as a regulator of gene expression in the vasculature
.
Circ Res
 
1999
;
85
:
753
766
.
7.
Ross
R
:
Atherosclerosis—an inflammatory disease
.
N Engl J Med
 
1999
;
340
:
115
126
.
8.
Kuller
LH
,
Tracy
RP
,
Shaten
J
,
Meilahn
EN
:
Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study. Multiple Risk Factor Intervention Trial
.
Am J Epidemiol
 
1996
;
144
:
537
547
.
9.
Blake
GJ
,
Rifai
N
,
Buring
JE
,
Ridker
PM
:
Blood pressure, C-reactive protein, and risk of future cardiovascular events
.
Circulation
 
2003
;
108
:
2993
2999
.
10.
Szmitko
PE
,
Wang
CH
,
Weisel
RD
,
de Almeida
JR
,
Anderson
TJ
,
Verma
S
:
New markers of inflammation and endothelial cell activation: part I
.
Circulation
 
2003
;
108
:
1917
1923
.
11.
Buono
C
,
Come
CE
,
Witztum
JL
,
Maguire
GF
,
Connelly
PW
,
Carroll
M
,
Lichtman
AH
:
Influence of C3 deficiency on atherosclerosis
.
Circulation
 
2002
;
105
:
3025
3031
.
12.
Rosenson
RS
,
Koenig
W
:
High-sensitivity CRP and cardiovascular risk in CHD patients
.
Curr Opin Cardiol
 
2002
;
17
:
325
331
.
13.
Verma
S
,
Wang
CH
,
Li
SH
,
Dumont
AS
,
Fedak
PW
,
Badiwala
MV
,
Dhillon
B
,
Weisel
RD
,
Li
RK
,
Mickle
DA
,
Stewart
DJ
:
A self-fulfilling prophecy. C-reactive protein attenuates nitric oxide production and inhibits angiogenesis
.
Circulation
 
2002
;
106
:
913
919
.
14.
Patrono
C
,
FitzGerald
GA
:
Isoprostanes: potential markers of oxidant stress in atherothrombotic disease
.
Arterioscler Thromb Vasc Biol
 
1997
;
17
:
2309
2315
.
15.
Deanfield
J
,
Donald
A
,
Ferri
C
,
Giannattasio
C
,
Halcox
J
,
Halligan
S
,
Lerman
A
,
Mancia
G
,
Oliver
JJ
,
Pessina
AC
,
Rizzoni
D
,
Rossi
GP
,
Salvetti
A
,
Schiffrin
EL
,
Taddei
S
,
Webb
DJ
,
Working Group on Endothelin and Endothelial Factors of the European Society of Hypertension
:
Endothelial function and dysfunction. Part I: methodological issues for assessment in the different vascular beds: a statement by the Working Group on endothelin and endothelial factors of the European Society of Hypertension
.
J Hypertension
 
2005
;
23
:
7
17
.
16.
Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure
:
The Seventh Report of The Joint National Committee, Evaluation and Treatment of High Blood Pressure
.
J Am Med Assoc
 
2003
;
157
:
2413
2446
.
17.
De Kossodo
S
,
WHO Collaborative Study Group
:
Assaying tumor necrosis factor concentration in human serum. A WHO International Collaborative Study
.
J Immunol Methods
 
1995
;
182
:
107
111
.
18.
Wang
CH
,
Li
SH
,
Weisel
RD
,
Fedak
PW
,
Dumont
AS
,
Szmitko
P
,
Li
RK
,
Mickle
DA
,
Verma
S
:
C-reactive protein upregulates angiotensin type 1 receptor in vascular smooth muscle
.
Circulation
 
2003
;
107
:
1783
1790
.
19.
Pasceri
V
,
Willerson
JT
,
Yeh
E
:
Direct proinflammatory effect of C-reactive protein on human endothelial cells
.
Circulation
 
2000
;
102
:
2165
2168
.
20.
Sesso
H
,
Buring
J
,
Rifai
N
,
Blage
G
,
Cazano
J
,
Ridker
PM
:
C reactive protein and risk for developing hypertension
.
J Am Med Assoc
 
2003
;
290
:
2945
2951
.
21.
Clausell
N
,
Kalil
P
,
Biolo
A
,
Molossi
S
,
Azevedo
M
:
Increased expression of tumor necrosis factor-α in diabetic macrovasculopathy
.
Cardiovasc Pathol
 
1999
;
8
:
145
151
.
22.
Waltenberger
J
,
Fitzsimmons
C
,
Hombach
V
:
C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries
.
Arterioscler Thromb Vasc Biol
 
1998
;
18
:
1386
1392
.
23.
Chen
X
,
Touyz
RM
,
Park
JB
,
Schiffrin
EL
:
Antioxidant effects of vitamins C and E are associated with altered activation of vascular NADPH oxidase and superoxide dismutase in stroke prone SHR
.
Hypertension
 
2001
;
38
:
606
611
.
24.
Taddei
S
,
Virdis
A
,
Ghiadoni
L
,
Magagna
A
,
Salvetti
A
:
Vitamin C improves endothelium-dependent vasodilation by restoring nitric oxide activity in essential hypertension
.
Circulation
 
1998
;
97
:
2222
2229
.
25.
Heitzer
T
,
Schlinzig
T
,
Krohn
K
,
Meinertz
T
,
Munzel
T
:
Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease
.
Circulation
 
2001
;
104
:
2673
2678
.
26.
Khan
BV
,
Harrison
DG
,
Olbrych
MT
,
Alexander
RW
,
Medford
RM
:
Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells
.
Proc Natl Acad Sci USA
 
1996
;
93
:
9114
9119
.
27.
Cracowski
JL
,
Baguet
JP
,
Ormezzano
O
,
Bessard
J
,
Stanke-Labesque
F
,
Bessard
G
,
Mallion
JM
:
Lipid peroxidation is not increased in patients with untreated mild-moderate hypertension
.
Hypertension
 
2003
;
41
:
286
288
.
28.
Ward
NC
,
Hodggson
JM
,
Puddey
IB
,
Mori
TA
,
Beilin
LJ
,
Croft
KD
:
Oxidative stress in human hypertension: association with antihypertensive treatment, gender, nutrition, and lifestyle
.
Free Radic Biol Med
 
2004
;
36
:
226
232
.
29.
Minuz
P
,
Patrignani
P
,
Gaino
S
,
Seta
F
,
Capone
ML
,
Tacconelli
S
,
Degan
M
,
Faccini
G
,
Fornasiero
A
,
Talamini
G
,
Tommasoli
R
,
Arosio
E
,
Santonastaso
CL
,
Lechi
A
,
Patrono
C
:
Determinants of platelet activation in human essential hypertension
.
Hypertension
 
2004
;
43
:
64
70
.
30.
Morrow
JD
:
Quantification of isoprostanes as indices of oxidant stress and risk of atherosclerosis in humans
.
Arterioscler Thromb Vasc Biol
 
2005
;
25
:
279
286
.