Increased levels of sCD40L (soluble CD40 ligand) have been associated with enhanced in vivo platelet activation, and may represent a molecular link between inflammation and a prothrombotic state. The aim of the present study was to analyse the relationship between platelet activation, endothelial dysfunction, low-grade inflammation and sCD40L in patients with hypertension with or without MA (microalbuminuria). A cross-sectional comparison of sCD40L levels was performed in 25 patients with MH (essential hypertension with MA) pair-matched for gender and age with 25 patients with EH (essential hypertension) and 25 HS (healthy subjects with normotension). Circulating levels of CRP (C-reactive protein), a marker of inflammation, sP-selectin (soluble P-selectin), a marker of in vivo platelet activation, and ADMA (asymmetric dimethylarginine) and vWF (von Willebrand factor), markers of endothelial dysfunction, were analysed in each subject. sCD40L levels were increased in patients with MH compared with either patients with EH (P<0.001) or HS (P<0.0001). A highly significant correlation between plasma sCD40L and sP-selectin (P<0.0001), vWF (P<0.001) or CRP levels (P<0.05) was observed in patients with MH. Multivariate regression analysis showed that sP-selectin was the strongest independent predictor of sCD40L levels (P<0.0001) in patients with MH. Patients with hypertension with both vWF and CRP levels above the median had the highest sCD40L levels (P<0.0001). Factorial ANOVA of all of the patients with hypertension confirmed that only patients with MH with low-grade inflammation had elevated levels of sCD40L. In conclusion, sCD40L levels appear to discriminate a subset of patients characterized by MA and low-grade inflammation, suggesting that inhibition of the CD40/CD40L system may represent a potential therapeutic target in subjects with hypertension at a high risk of cardiovascular events.

INTRODUCTION

CD40L (CD40 ligand) is a transmembrane protein, structurally related to TNF-α (tumour necrosis factor-α), that was first identified on CD4+ T-cells [1,2]. Subsequently, CD40L has been found on the surface membrane of activated platelets [3] from where sCD40L (soluble CD40L) can be released into the circulation [4]. Both membrane-bound CD40L and sCD40L interact with CD40 expressed on vascular cells, resulting in inflammatory and prothrombotic responses [3,5]. Engagement of CD40L with CD40 on endothelial cells, in fact, results in phenotypic changes that are similar to those induced by TNF-α, i.e. increased expression of adhesion molecules and secretion of inflammatory cytokines [6]. In addition, sCD40L can act as a ligand of the platelet fibrinogen receptor glycoprotein IIb/IIIa, and is involved in thrombus stabilization and platelet activation [7]. Thus sCD40L may represent a key molecule linking inflammation and a prothrombotic state in human diseases.

To date, it is estimated that more than 95% of plasma sCD40L is derived from platelets [8]. Accordingly, increased levels of sCD40L have been associated with enhanced in vivo platelet activation in various clinical conditions [915]. Meanwhile, it has become evident that sCD40L could be regarded as a predictive marker of cardiovascular events in healthy individuals [15,16], which may not only indicate active inflammation and platelet activation, but may also exhibit direct prothrombotic properties that mediate early atherogenesis, plaque rupture and thrombosis [17].

Persistent platelet activation has been demonstrated in patients with essential hypertension, especially those with advanced vascular lesions [1821], and strict BP (blood pressure) control may result in significant improvements in platelet function in subjects with hypertension at high risk of cardiovascular events [22]. In addition, low-grade inflammation [23,24], as shown by increased levels of CRP (C-reactive protein), and endothelial dysfunction [2427], as shown by increased levels of vWF (von Willebrand factor) or ADMA (asymmetric dimethylarginine), have been frequently associated with essential hypertension and may help to identify patients at very high cardiovascular risk.

Although several factors link platelet activation, endothelial dysfunction and low-grade inflammation with sCD40L, no information is available about their inter-relationships with hypertension-related cardiovascular and metabolic abnormalities. Therefore in the present study we investigated this issue in patients with hypertension with or without MA (microalbuminuria), which is currently acknowledged as a predictor of vascular disease [28] and the strongest independent determinant of ischaemic heart disease among non-diabetic individuals with arterial hypertension [29,30].

MATERIALS AND METHODS

Design of the study

A cross-sectional comparison was performed in 50 patients with EH (essential hypertension without MA, i.e. normomicroalbuminuric) or MH (essential hypertension with MA) and in 25 HS (healthy subjects with normotension) enrolled at our Institutions (Table 1). Patients with MH (n=25) were pair-matched for gender and age with patients with EH (n=25) and HS (Table 1).

Table 1
Baseline characteristics of HS, and patients with EH and MH

Values are means±S.D. or medians (25–75th percentile). *P<0.0001 compared with HS; †P<0.0001 compared with patients with EH. For AER, H=62.7, P<0.0001 using the Kruskal–Wallis test.

VariableHSPatients with EHPatients with MH
n 25 25 25 
Male gender (n12 (48%) 12 (48%) 12 (48%) 
Age (years) 66.5±12.5 69.8±10.9 69.4±12.1 
BMI (kg/m224.9±2.2 26.2±2.1 24.9±2.9 
Disease duration (years) — 12.5±8.7 9.0±7.9 
Systolic BP (mmHg) 125±8.0 161±18.7 155±8.5 
Diastolic BP (mmHg) 74±9.0 94±12.0 90±6.8 
LDL-cholesterol (mg/dl) 117±29 127±26 113±31 
HDL-cholesterol (mg/dl) 42±5 46±8 42±8 
Triacylglycerols (mg/dl) 105±40 117±34 137±38 
AER (μg/min) 5.2 (4.1–5.9) 11.3 (8.7–12.5)* 86.8 (50.0–176.0)*† 
Creatinine (mg/dl) 0.95±0.19 0.95±0.19 0.92±0.21 
VariableHSPatients with EHPatients with MH
n 25 25 25 
Male gender (n12 (48%) 12 (48%) 12 (48%) 
Age (years) 66.5±12.5 69.8±10.9 69.4±12.1 
BMI (kg/m224.9±2.2 26.2±2.1 24.9±2.9 
Disease duration (years) — 12.5±8.7 9.0±7.9 
Systolic BP (mmHg) 125±8.0 161±18.7 155±8.5 
Diastolic BP (mmHg) 74±9.0 94±12.0 90±6.8 
LDL-cholesterol (mg/dl) 117±29 127±26 113±31 
HDL-cholesterol (mg/dl) 42±5 46±8 42±8 
Triacylglycerols (mg/dl) 105±40 117±34 137±38 
AER (μg/min) 5.2 (4.1–5.9) 11.3 (8.7–12.5)* 86.8 (50.0–176.0)*† 
Creatinine (mg/dl) 0.95±0.19 0.95±0.19 0.92±0.21 

Exclusion criteria included a history or evidence of atherothrombotic diseases, diabetes mellitus, cigarette smoking, dyslipidaemia, obesity and the metabolic syndrome in order to avoid confounding with other determinants of endothelial dysfunction and platelet activation. None of the subjects was permitted to take vitamin supplements, NSAIDs (non-steroidal antiinflammatory drugs) or antiplatelet agents for at least 30 days before the study. Women who were taking hormonal replacement therapy were also excluded.

Essential hypertension was diagnosed clinically according to the WHO (World Health Organization) criteria [31] on three consecutive occasions and on the basis of negative results of investigation for secondary hypertension, which included hormonal evaluation, renal scintigraphy and/or echo-Doppler scan of the renal arteries. MA was defined as a urinary AER (albumin excretion rate) between 20 and 200 μg/min. The patients with hypertension were under long-term treatment with antihypertensive medication (angiotensin-converting-enzyme inhibitors and/or diuretics and/or calcium antagonists). Both groups of patients with hypertension received a standard diet containing approx. 150 mmol of sodium/day. The mean duration of hypertension was defined as the time between the diagnosis and inclusion in the present study.

All subjects were studied on an outpatient basis. Blood samples were obtained in the early morning after a 12-h fast.

The study was performed after approval from the Ethics Committee of the Civil Hospital, Pescara, Italy, and in accordance with the principles embodied in the Declaration of Helsinki. Informed consent was obtained from each participating subject.

Biochemical analyses

The mean AER was calculated from three consecutive timed overnight urine collections. AER was measured by nephelometry (Roche Diagnostics). Serum CRP levels were measured with a highly sensitive immunoassay [32]. Total cholesterol, triacylglycerols (triglycerides), HDL (high-density lipoprotein)-cholesterol and LDL (low-density lipoprotein)-cholesterol concentrations were measured as described previously [18].

Plasma sP-selectin (soluble P-selectin), sCD40L and vWF levels were measured in citrated plasma samples by commercially available ELISAs (from R&D Systems for sP-selectin and sCD40L, and American Diagnostica for vWF), according to the manufacturers' instructions. ADMA levels were measured in plasma samples by a commercially available enzyme-immunometric assay (DLD Diagnostika), validated previously by comparison with GC/MS [33]. Plasma samples were processed promptly after collection (within 2 h) to minimize ex vivo release of P-selectin and CD40L from platelets [34].

Statistical analysis

Statistical analysis was performed by χ2 statistics, Pearson's correlation coefficient and by one-way ANOVA with Bonferroni adjustment to assess differences between the groups (patients with EH, patients with MH and HS). Where necessary, appropriate non-parametric tests were employed (Spearman's correlation coefficient, Kruskal–Wallis method and Mann–Whitney U test). Multiple linear regression analysis and factorial ANOVA were performed to analyse the data further. Multiple regression analysis always included, as the independent variables, male gender, age, BMI (body mass index), AER, disease duration, antihypertensive treatment, systolic and diastolic BP, blood lipids and smoking habit. With 25 subjects recruited in the cross-sectional comparison, the study had a 95% power to detect a 40% difference in sCD40L levels between the patients with MH, patients with EH and HS with a two-tailed α of 0.05. Results are presented as means (±S.D.) or medians (25–75th percentile). Only P values <0.05 were regarded as statistically significant. All tests were two-tailed, and analyses were performed using a computer software package [either Statistica 6.0 (StatSoft) or SPSS version 13.0].

RESULTS

Baseline characteristics of the three groups of subjects are shown in Table 1. Patients with EH or MH did not differ in terms of BP, disease duration, creatinine serum level and serum lipid pattern. HS had similar clinical and laboratory characteristics compared with patients with EH or MH. Urinary AER was significantly higher in patients with MH compared with patients with EH and HS (P<0.0001); patients with EH had significantly higher values of urinary AER than HS (P<0.0001).

Platelet activation, as reflected by sP-selectin levels, was significantly enhanced in patients with MH compared with either HS or patients with EH (Table 2). Furthermore, an impairment of endothelial function was found in patients with either EH or MH, as shown by increased plasma levels of both vWF and ADMA compared with HS (Table 2). Elevated ADMA (r=0.259, P=0.01) or vWF (r=0.595, P<0.0001) levels, low HDL-cholesterol (r=−0.305, P=0.001) and disease duration (r=0.244, P=0.004) were all predictive of MA by multivariate analysis.

Table 2
Laboratory variables of HS, and patients with EH and MH

Values are means±S.D. or medians (25–75th percentile). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 compared with HS; †P<0.01 and ††P<0.0001 compared with patients with EH. ‡Obtained by either Kruskal–Wallis test (H) or ANOVA (F).

VariableHSPatients with EHPatients with MHTest statistic‡
n 25 25 25  
sCD40L (ng/ml) 0.7 (0.5–0.8) 0.9 (0.6–1.0) 1.6 (1.0–2.6)****† H=14.2, P<0.001 
sP-selectin (ng/ml) 68 (60–75) 86 (65–98)** 136 (122–176)****†† H=34.0, P<0.0001 
ADMA (μmol/l) 0.39 (0.34–0.56) 0.56 (0.46–0.75)*** 1.12 (0.76–1.32)****†† H=38.8, P<0.0001 
vWF (IU/dl) 728±133 892±148* 1447±292***† F=85.4, P<0.0001 
CRP (mg/dl) 0.36 (0.30–0.50) 0.90 (0.58–1.22)**** 1.6 (1.2–2.1)****† H=45.0, P<0.0001 
VariableHSPatients with EHPatients with MHTest statistic‡
n 25 25 25  
sCD40L (ng/ml) 0.7 (0.5–0.8) 0.9 (0.6–1.0) 1.6 (1.0–2.6)****† H=14.2, P<0.001 
sP-selectin (ng/ml) 68 (60–75) 86 (65–98)** 136 (122–176)****†† H=34.0, P<0.0001 
ADMA (μmol/l) 0.39 (0.34–0.56) 0.56 (0.46–0.75)*** 1.12 (0.76–1.32)****†† H=38.8, P<0.0001 
vWF (IU/dl) 728±133 892±148* 1447±292***† F=85.4, P<0.0001 
CRP (mg/dl) 0.36 (0.30–0.50) 0.90 (0.58–1.22)**** 1.6 (1.2–2.1)****† H=45.0, P<0.0001 

Plasma sCD40L levels were significantly increased in patients with MH compared with either patients with EH or HS (Table 2 and Figure 1), but no differences were observed between the two latter groups (Figure 1). A highly significant correlation between plasma sCD40L and sP-selectin (ρ=0.750, P<0.0001), vWF (ρ=0.658, P<0.0001) or CRP levels (ρ=0.42, P<0.003) was observed in patients with hypertension. Subgroup analysis revealed that these associations were retained in the subgroup of patients with MH (Figure 2), but not in patients with EH, in whom only the correlation between sCD40L and sP-selectin (ρ=0.528, P<0.007) was retained. Stepwise multivariate regression analysis showed that sP-selectin was the strongest independent predictor of sCD40L levels [b (standard error)=0.787 (0.13), P<0.0001] in patients with MH.

Box-plot analysis of plasma sCD40L levels in patients with MH or EH compared with HS

Figure 1
Box-plot analysis of plasma sCD40L levels in patients with MH or EH compared with HS

Solid lines indicate median values, whiskers indicate the non-outlier range, and the boxes indicate 25–75th percentiles.

Figure 1
Box-plot analysis of plasma sCD40L levels in patients with MH or EH compared with HS

Solid lines indicate median values, whiskers indicate the non-outlier range, and the boxes indicate 25–75th percentiles.

Correlation analysis of plasma sCD40L levels and sP-selectin (top panel), vWF (middle panel) and CRP (bottom panel) in patients with MH

Figure 2
Correlation analysis of plasma sCD40L levels and sP-selectin (top panel), vWF (middle panel) and CRP (bottom panel) in patients with MH
Figure 2
Correlation analysis of plasma sCD40L levels and sP-selectin (top panel), vWF (middle panel) and CRP (bottom panel) in patients with MH

To evaluate further the relationship between sCD40L and endothelial dysfunction or inflammatory status, patients with hypertension were stratified on the basis of the observed median CRP (1.22 mg/dl) or vWF (1120 IU/dl) levels. As shown in Figure 3, patients with hypertension with both vWF and CRP levels above the median had the highest sCD40L levels (median level, 2.4 pg/ml) compared with patients with either vWF or CRP levels above the median (1.1 pg/ml), or patients with both variables below the median (0.65 pg/ml; H=21.5, P<0.0001). These results were confirmed in the subset of patients with MH (H=10.7, P<0.005), but not in patients with EH.

Box-plot analysis of plasma sCD40L levels in all patients with hypertension (n=50) with vWF and/or CRP levels below (negative) or above (positive) the median values

Figure 3
Box-plot analysis of plasma sCD40L levels in all patients with hypertension (n=50) with vWF and/or CRP levels below (negative) or above (positive) the median values
Figure 3
Box-plot analysis of plasma sCD40L levels in all patients with hypertension (n=50) with vWF and/or CRP levels below (negative) or above (positive) the median values

On the basis on these results, we finally performed a factorial ANOVA including data from all of the patients with hypertension to characterize better the interactions between sCD40L and the inflammatory status in the presence or absence of MA. The results in Figure 4 show that only patients with MH with low-grade inflammation had increased levels of sCD40L. Multivariate regression analysis of this restricted subset of patients confirmed the independent prognostic role of sP-selectin in determining sCD40L levels [b (standard error)=0.838 (0.15), P<0.0001].

Factorial ANOVA of plasma sCD40L levels in patients with MH or EH stratified on the basis of CRP levels below (negative) or above (positive) the median value

Figure 4
Factorial ANOVA of plasma sCD40L levels in patients with MH or EH stratified on the basis of CRP levels below (negative) or above (positive) the median value

Dots indicate median values, whiskers indicate the non-outlier range, and the boxes indicate 25–75th percentiles.

Figure 4
Factorial ANOVA of plasma sCD40L levels in patients with MH or EH stratified on the basis of CRP levels below (negative) or above (positive) the median value

Dots indicate median values, whiskers indicate the non-outlier range, and the boxes indicate 25–75th percentiles.

DISCUSSION

To date, no clinical evidence is available linking platelet activation and/or inflammation to sCD40L production in hypertension. The present study is the first to demonstrate that increased levels of this cytokine may be, at least in part, related to the occurrence of in vivo platelet activation, as demonstrated by the significant association found between sCD40L and sP-selectin, a well recognized marker of in vivo platelet activation [3537].

The occurrence of elevated sCD40L levels in patients with hypertension is still a debated issue. In fact, Tsioufis et al. [38] recently reported that MA is not accompanied by an increase in sCD40L concentrations in subjects with essential hypertension, and hypothesized the activation of different inflammatory pathways in the progression of renal and cardiovascular atherosclerotic disease in these patients. However, the study group used in that study was restricted to non-smoking subjects with hypertension without other risk factors (i.e. obesity). As Bautista [39] pointed out in his critique of the study by Tsioufis et al. [38], although selection is sometimes used to prevent confounding, it limits generalizability and is rarely justifiable, as confounding can be controlled efficiently using multiple regression analysis. In our cohort of patients in the present study, sCD40L levels were higher in patients with MH compared with patients with EH and age- and gender-matched HS. This finding is in agreement with those by others, suggesting that plasma sCD40L levels may contribute to the increased susceptibility of patients with hypertension in developing vascular damage [40], probably through blunted angiogenesis [41]. In particular, Patel et al. [41] suggested that sCD40L might reflect abnormal platelet activation and, thus, increased sCD40L (as a marker of CD40 ligation) may contribute to the increased cardiovascular risk in patients with hypertension.

In the present study, the findings of increased sCD40L levels were limited to a subset of patients with hypertension, namely those with MA. The latter significantly correlated with the duration of hypertension, which is in agreement with the findings of others [28]. Moreover, we have shown that increased plasma levels of vWF and ADMA (two well-recognized markers of endothelial dysfunction) were both predictive of MA. These findings confirm those reported in previous studies [2428] and are in agreement with those by Pedrinelli et al. [25], who demonstrated the presence of increased vWF levels in patients with essential hypertension and MA as well as intra-individual correlation between the two variables, suggesting that a dysfunctional glomerular endothelium may cause reduced renal function. Along with the occurrence of endothelial dysfunction (as shown by increased levels of both vWF and ADMA), in the present study, we observed a low-grade inflammatory condition in all patients with hypertension either with or without MA. Indeed, circulating CRP levels were significantly higher in patients with MH compared with patients with EH or HS. This is not surprising if we consider a recent study by Sesso et al. [23], who demonstrated that CRP levels are associated with the future development of hypertension, which suggests that hypertension is in part an inflammatory disorder.

The major conclusion that may be drawn from the present study is that sCD40L levels appear to discriminate a high-risk subset of patients characterized by MA and low-grade inflammation. This is of particular interest as several population-based studies have shown an association between MA and cardiovascular morbidity and mortality [28], leading to the demonstration that MA is the strongest independent determinant of ischaemic heart disease in subjects with arterial hypertension [29]. Furthermore, MA accompanied by evidence of subclinical inflammation has been proposed as a strong correlate of metabolic abnormalities in essential hypertension and might identify a subset of patients at very high cardiovascular risk. In contrast, isolated MA may represent a distinct pathophysiological condition characterized by a more benign profile and possibly a better prognosis [24]. In this context, the finding of increased levels of sCD40L mainly in a subgroup of patients with MH with low-grade inflammation (as shown by elevated CRP concentrations) suggests that this cytokine might be directly responsible for prothrombotic properties that mediate thrombosis and acute vascular events.

One limitation of the present study is the small number of patients; however, we must consider that the population enrolled was highly selected, as we excluded patients with hypertension with concomitant risk factors for atherothrombosis, such as obesity, cigarette smoking, diabetes mellitus and the metabolic syndrome, all of which have been shown to significantly affect the levels of urinary albumin excretion and to be associated with biochemical evidence of in vivo platelet activation [913,15,42,43]. In terms of a study limitation, the present findings were limited to patients with MH, a surrogate of the subclinical atherosclerotic burden and low-grade inflammation, limiting the generalization of the study. Nevertheless, the present study may provide new insights into the pathophysiological mechanisms linking early renal impairment with elevated cardiovascular risk.

In conclusion, the results of the present study confirm that CD40L represents a key molecule linking inflammation and thrombosis. Thus inhibition of the CD40/CD40L system may represent a potential therapeutic target in a subgroup of patients with hypertension at high risk of cardiovascular events.

Abbreviations

     
  • ADMA

    asymmetric dimethylarginine

  •  
  • AER

    albumin excretion rate

  •  
  • BMI

    body mass index

  •  
  • BP

    blood pressure

  •  
  • CD40L

    CD40 ligand

  •  
  • CRP

    C-reactive protein

  •  
  • EH

    essential hypertension

  •  
  • HDL

    high-density lipoprotein

  •  
  • HS

    healthy subjects with normotension

  •  
  • LDL

    low-density lipoprotein

  •  
  • MA

    microalbuminuria

  •  
  • EH

    essential hypertension without MA

  •  
  • MH

    essential hypertension with MA

  •  
  • sCD40L

    soluble CD40L

  •  
  • sP-selectin

    soluble P-selectin

  •  
  • TNF-α

    tumour necrosis factor-α

  •  
  • vWF

    von Willebrand factor

The study was supported by the Italian Ministry of University and Research (Prin2006063532) to G.D.

References

References
1
Mach
 
F.
Schonbeck
 
U.
Sukhova
 
G. K.
, et al 
Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implication for CD40-CD40 ligand signaling in atherosclerosis
Proc. Natl. Acad. Sci. U.S.A.
1997
, vol. 
94
 (pg. 
1931
-
1936
)
2
van Kooten
 
C.
 
Immune regulation by CD40-CD40L interactions
Front. Biosci.
2000
, vol. 
5
 (pg. 
680
-
693
)
3
Henn
 
V.
Slupsky
 
J. R.
Grafe
 
M.
, et al 
CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells
Nature
1998
, vol. 
391
 (pg. 
591
-
594
)
4
Aukrust
 
P.
Muller
 
F.
Ueland
 
T.
, et al 
Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina. Possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes
Circulation
1999
, vol. 
100
 (pg. 
614
-
620
)
5
Lindmark
 
E.
Tenno
 
T.
Siegbahn
 
A.
 
Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression
Arterioscler. Thromb. Vasc. Biol.
2000
, vol. 
20
 (pg. 
2322
-
2328
)
6
Granger
 
D. N.
Vowinkel
 
T.
Petnehazy
 
T.
 
Modulation of the inflammatory response in cardiovascular disease
Hypertension
2004
, vol. 
43
 (pg. 
924
-
931
)
7
Prasad
 
K. S.
Andre
 
P.
Yan
 
Y.
Phillips
 
D. R.
 
The platelet CD40L/GP IIb-IIIa axis in atherothrombotic disease
Curr. Opin. Hematol.
2003
, vol. 
10
 (pg. 
356
-
361
)
8
Freedman
 
J. E.
 
CD40-CD40L and platelet function
Beyond hemostasis. Circ. Res.
2003
, vol. 
92
 (pg. 
944
-
946
)
9
Cipollone
 
F.
Mezzetti
 
A.
Porreca
 
E.
, et al 
Association between enhanced soluble CD40L and prothrombotic state in hypercholesterolemia: effects of statin therapy
Circulation
2002
, vol. 
106
 (pg. 
399
-
402
)
10
Garlichs
 
C. D.
Kozina
 
S.
Fateh-Moghadam
 
S.
, et al 
Upregulation of CD40-CD40 ligand (CD154) in patients with acute cerebral ischemia
Stroke
2003
, vol. 
34
 (pg. 
1412
-
1418
)
11
Danese
 
S.
Katz
 
J. A.
Saibeni
 
S.
, et al 
Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients
Gut
2003
, vol. 
52
 (pg. 
1435
-
1441
)
12
Lim
 
H. S.
Blann
 
A. D.
Lip
 
G. Y.
 
Soluble CD40 ligand, soluble P-selectin, interleukin-6, and tissue factor in diabetes mellitus: relationships to cardiovascular disease and risk factor intervention
Circulation
2004
, vol. 
109
 (pg. 
2524
-
2528
)
13
Falco
 
A.
Romano
 
M.
Iapichino
 
L.
Collura
 
M.
Davi
 
G.
 
Increased soluble CD40 ligand levels in cystic fibrosis
J. Thromb. Haemostasis
2004
, vol. 
2
 (pg. 
557
-
560
)
14
Roselli
 
M.
Mineo
 
T. C.
Basili
 
S.
, et al 
Soluble CD40 ligand plasma levels in lung cancer
Clin. Cancer Res.
2004
, vol. 
10
 (pg. 
610
-
614
)
15
Novo
 
S.
Basili
 
S.
Tantillo
 
R.
, et al 
Soluble CD40L and cardiovascular risk in asymptomatic low-grade carotid stenosis
Stroke
2005
, vol. 
36
 (pg. 
673
-
675
)
16
Schonbeck
 
U.
Varo
 
N.
Libby
 
P.
Buring
 
J.
Ridker
 
P. M.
 
Soluble CD40L and cardiovascular risk in women
Circulation
2001
, vol. 
104
 (pg. 
2266
-
2268
)
17
Mo
 
V. Y.
De Lemos
 
J. A.
 
Individualizing therapy in acute coronary syndromes: using a multiple biomarker approach for diagnosis, risk stratification, and guidance of therapy
Curr. Cardiol. Rep.
2004
, vol. 
6
 (pg. 
273
-
278
)
18
Davì
 
G.
Gresele
 
P.
Violi
 
F.
, et al 
Diabetes mellitus, hypercholesterolemia, and hypertension but not vascular disease per se are associated with persistent platelet activation in vivo. Evidence derived from the study of peripheral arterial disease
Circulation
1997
, vol. 
96
 (pg. 
69
-
75
)
19
Spencer
 
C. G.
Gurney
 
D.
Blann
 
A. D.
Beevers
 
D. G.
Lip
 
G. Y.
 
Von Willebrand factor, soluble P-selectin, and target organ damage in hypertension: a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT)
Hypertension
2002
, vol. 
40
 (pg. 
61
-
66
)
20
Minuz
 
P.
Patrignani
 
P.
Gaino
 
S.
, et al 
Determinants of platelet activation in human essential hypertension
Hypertension
2004
, vol. 
43
 (pg. 
64
-
70
)
21
Nadar
 
S. K.
Blann
 
A. D.
Kamath
 
S.
Beevers
 
D. G.
Lip
 
G. Y. H.
 
Platelet indexes in relation to target organ damage in high-risk hypertensive patients: a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT)
J. Am. Coll. Cardiol.
2004
, vol. 
44
 (pg. 
415
-
422
)
22
Blann
 
A. D.
Nadar
 
S.
Lip
 
G. Y.
 
Pharmacological modulation of platelet function in hypertension
Hypertension
2003
, vol. 
42
 (pg. 
1
-
7
)
23
Sesso
 
H. D.
Buring
 
J. E.
Rifai
 
N.
Blake
 
G. J.
Gaziano
 
J. M.
Ridker
 
P. M.
 
C-reactive protein and the risk of developing hypertension
JAMA, J. Am. Med. Assoc.
2003
, vol. 
290
 (pg. 
2945
-
2951
)
24
Pedrinelli
 
R.
Dell'Omo
 
G.
Di Bello
 
V.
, et al 
Low-grade inflammation and microalbuminuria in hypertension
Arterioscler. Thromb. Vasc. Biol.
2004
, vol. 
24
 (pg. 
2414
-
2419
)
25
Pedrinelli
 
R.
Giampietro
 
O.
Carmassi
 
F.
, et al 
Microalbuminuria and endothelial dysfunction in essential hypertension
Lancet
1994
, vol. 
344
 (pg. 
14
-
18
)
26
Taddei
 
S.
Salvetti
 
A.
 
Endothelial dysfunction in essential hypertension: clinical implications
J. Hypertens.
2002
, vol. 
20
 (pg. 
1671
-
1674
)
27
Goonasekera
 
C. D.
Shah
 
V.
Rees
 
D. D.
Dillon
 
M. J.
 
Vascular endothelial cell activation associated with increased plasma asymmetric dimethyl arginine in children and young adults with hypertension: a basis for atheroma?
Blood Press.
2000
, vol. 
9
 (pg. 
16
-
21
)
28
Garg
 
J. P.
Bakris
 
G. L.
 
Microalbuminuria: marker of vascular dysfunction, risk factor for cardiovascular disease
Vasc. Med.
2002
, vol. 
7
 (pg. 
35
-
43
)
29
Jensen
 
J. S.
Feldt-Rasmussen
 
B.
Strandgaard
 
S.
Schroll
 
M.
Borch-Johnsen
 
K.
 
Arterial hypertension, microalbuminuria, and risk of ischemic heart disease
Hypertension
2000
, vol. 
35
 (pg. 
898
-
903
)
30
Jager
 
A.
Kostense
 
P. J.
Ruhe
 
H. G.
, et al 
Microalbuminuria and peripheral arterial disease are independent predictors of cardiovascular and all-cause mortality, especially among hypertensive subjects: five-year follow-up of the Hoorn Study
Arterioscler. Thromb. Vasc. Biol.
1999
, vol. 
19
 (pg. 
617
-
624
)
31
World Health Organization-International Society of Hypertension Guidelines for the Management of Hypertension
Guidelines Subcommittee
J. Hypertens.
1999
, vol. 
17
 (pg. 
151
-
183
)
32
Ledue
 
T. B.
Weiner
 
D. L.
Sipe
 
J. D.
Poulin
 
S. E.
Collins
 
M. F.
Rifai
 
N.
 
Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid A and mannose-binding protein in human serum
Ann. Clin. Biochem.
1998
, vol. 
35
 (pg. 
745
-
753
)
33
Schulze
 
F.
Wesemann
 
R.
Schwedhelm
 
E.
, et al 
Determination of asymmetric dimethylarginine (ADMA) using a novel ELISA assay
Clin. Chem. Lab. Med.
2004
, vol. 
42
 (pg. 
1377
-
1383
)
34
Thom
 
J.
Gilmore
 
G.
Yi
 
Q.
Hankey
 
G. J.
Eikelboom
 
J. W.
 
Measurement of soluble P-selectin and soluble CD40 ligand in serum and plasma
J. Thromb. Haemostasis
2004
, vol. 
2
 (pg. 
2067
-
2069
)
35
Blann
 
A. D.
Nadar
 
S. K.
Lip
 
G. Y.
 
The adhesion molecule P-selectin and cardiovascular disease
Eur. Heart J.
2003
, vol. 
24
 (pg. 
2166
-
2179
)
36
Davì
 
G.
Romano
 
M.
Mezzetti
 
A.
, et al 
Increased levels of soluble P-selectin in hypercholesterolemic patients
Circulation.
1998
, vol. 
97
 (pg. 
953
-
957
)
37
Andre
 
P.
 
P-selectin in haemostasis
Br. J. Haematol.
2004
, vol. 
126
 (pg. 
298
-
306
)
38
Tsioufis
 
C.
Dimitriadis
 
K.
Taxiarchou
 
E.
, et al 
Diverse associations of microalbuminuria with C-reactive protein, interleukin-18 and soluble CD40 ligand in male essential hypertensive subjects
Am. J. Hypertens.
2006
, vol. 
19
 (pg. 
462
-
466
)
39
Bautista
 
L. E.
 
Associations of microalbuminuria with inflammation markers in hypertensive men
Am. J. Hypertens.
2006
, vol. 
19
 pg. 
467
 
40
Desideri
 
G.
Cipollone
 
F.
Valeri
 
L.
, et al 
Enhanced plasma soluble CD40 ligand levels in essential hypertensive patients with blunted nocturnal blood pressure decrease
Am. J. Hypertens.
2007
, vol. 
20
 (pg. 
70
-
76
)
41
Patel
 
J. V.
Lim
 
H. S.
Nadar
 
S.
Tayebjee
 
M.
Hughes
 
E. A.
Lip
 
G. Y.
 
Abnormal soluble CD40 ligand and C-reactive protein concentrations in hypertension: relationship to indices of angiogenesis
J. Hypertens.
2006
, vol. 
24
 (pg. 
117
-
121
)
42
Lee
 
W. L.
Lee
 
W. J.
Chen
 
Y. T.
, et al 
The presence of metabolic syndrome is independently associated with elevated serum CD40 ligand and disease severity in patients with symptomatic coronary artery disease
Metab., Clin. Exp.
2006
, vol. 
55
 (pg. 
1029
-
1034
)
43
Angelico
 
F.
Alessandri
 
C.
Ferro
 
D.
, et al 
Enhanced soluble CD40L in patients with the metabolic syndrome: relationship with in vivo thrombin generation
Diabetologia
2006
, vol. 
49
 (pg. 
1169
-
1174
)