This Article Abstract Full Text (PDF) All Versions of this Article: 36/3/673    most recent 01.STR.0000154878.58398.14v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Novo, S. Articles by Davì, G. Search for Related Content PubMed PubMed Citation Articles by Novo, S. Articles by Davì, G. Pubmed/NCBI databases Substance via MeSH Medline Plus Health Information Stroke Related Collections Risk Factors Carotid Stenosis Doppler ultrasound, Transcranial Doppler etc.

(Stroke. 2005;36:673.)
© 2005 American Heart Association, Inc.

Research Report

Soluble CD40L and Cardiovascular Risk in Asymptomatic Low-Grade Carotid Stenosis

Salvatore Novo, MD; Stefania Basili, MD; Rosalba Tantillo, MD; Angela Falco, MD; Valentina Davì, MD; Giuseppina Novo, MD; Egle Corrado, MD Giovanni Davì, MD

From the Department of Clinical Medicine and Emerging Pathologies (S.N., R.T., V.D., G.N., C.E.), University of Palermo, Italy; the Department of Medical Therapy (S.B.), University of Rome "La Sapienza," Italy; and the Center of Excellence on Aging (A.F., G.D.), "G. d’Annunzio" University Foundation, University of Chieti, Italy.

Correspondence to Dr Giovanni Davì, "G. d’Annunzio" University Foundation, University of Chieti School of Medicine, Via Colle dell’Ara, 66013 Chieti, Italy. E-mail gdavi{at}unich.it

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose— We investigated whether soluble CD40L (sCD40L) may predict the risk of cardiovascular (CV) events in patients with asymptomatic carotid plaques.

Methods— Forty-two patients with asymptomatic low-grade carotid stenosis (ALCS) and 21 controls without any carotid stenosis were enrolled. All subjects had at least a major cardiovascular risk factor (CRF). Plasma levels of C-reactive protein (CRP), IL-6, and sCD40L were measured. Subjects were reviewed every 12 months (median follow-up, 8 years).

Results— ALCS patients had higher (P<0.0001) CRP, IL-6, and sCD40L than controls. Fourteen patients experienced a CV event. Cox regression analysis showed that only high sCD40L levels (P=0.003) independently predicted cardiovascular risk.

Conclusions— High levels of sCD40L may predict the risk of CV events in ALCS.

Key Words: atherosclerosis • carotid stenosis • inflammation • risk factors

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Carotid artery intima media thickness is associated with cardiovascular diseases (CVD).¹ Studies support the role of CD40–CD40 ligand interactions in atherothrombosis.² Patients with acute cerebral ischemia show upregulation of CD40 system.³

Cytokines and cytokine-inducible inflammatory molecules are regarded as predictive markers of cardiovascular events. Enhanced levels of C-reactive protein (CRP), IL-6, and soluble CD40 ligand (sCD40L) are associated with high risk of CVD.^4,5 We investigated whether these measurements may predict cardiovascular events in asymptomatic carotid plaques patients, beyond the current "standard" risk factors (CRF).

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects without previous CVD were examined for carotid atherosclerotic involvement because of presence of at least one CRF. Intima media thickness >1.5 mm, acute and chronic inflammatory diseases, treatment with hormonal replacement therapy, steroids, aspirin, nonsteroidal anti-inflammatory drugs, or antioxidant supplements represented exclusion criteria.

Forty-two patients with asymptomatic low-grade carotid stenosis (ALCS) and 21 subjects without any carotid stenosis were consecutively enrolled (Table 1). All gave written informed consent. The study was approved by local ethics committee.

View this table: [in this window] [in a new window]   TABLE 1. Subject Characteristics

Patients were reviewed every 12 months or when they experienced cardiovascular events (myocardial infarction, angina, stroke, transient ischemic attack, carotid endarterectomy, intermittent claudication, need for percutaneous revascularization procedures or coronary artery bypass graft) (median follow-up, 8 years).

Blood samples were anticoagulated in Na citrate 3.8% (1:9 v:v), centrifuged at 1500g for 10 minutes, and plasma was stored at –80°C until analysis.

IL-6 (R&D Systems) and sCD40L (Bender Medsystems) were measured by enzyme-linked immunosorbent assay; CRP by highly sensitive immunoassay (Abbot). Intra-assay and interassay coefficients of variation were <9%.

The carotid arteries were evaluated with high-resolution B-mode ultrasonography using a 7.5-MHz duplex-type probe (Toshiba). Percent stenosis was graded as low-grade lumen stenosis because of plaque >15% but <50% (intima media thickness >0.85 and <1.5 mm). If no lesion was detected, subjects were considered as normal.

Statistical Analysis
Statistical analysis was performed by ² statistics, Pearson correlation coefficient, and by t test for independent samples. When necessary, appropriate nonparametric tests were used (Spearman correlation coefficient and Mann–Whitney U test). A multiple linear regression analysis was performed to further quantify the relationship between sCD40L and the variables in study. The clinical relevance of parameters for the prediction of all events and separately of combined endpoints myocardial infarction and stroke (hard endpoints) were estimated by univariate (by Log Rank Test) and multivariate (by Cox proportional hazard model including variables that achieved statistical significance in the univariate analysis) analyses.

Data are presented as mean (SD) or median (range) (SPSS by SPSS Inc and EGRET by SERC).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Patients and controls displayed comparable body mass index and lipid levels. In contrast, the prevalence of hypertension and diabetes was increased in ALCS (Table 1). ALCS had higher (P<0.0001) CRP, IL-6, and sCD40L than controls (Table 1). This difference was still significant after patients’ stratification according to CRF presence, mainly hypertension and diabetes.

Among ALCS, sCD40L directly correlated with CRP (R_s=0.59; P<0.0001) and IL-6 (R_s=0.63; P<0.0001). Multiple regression analysis indicated that IL-6 (ß=0.41, SE=0.16; P<0.02) and CRP (ß=0.36, SE=0.15; P<0.03) predicted sCD40L levels, independently of CRF.

Median follow-up was 3225 (37 to 3681) days. No patient was lost to follow-up. Fourteen (33%) patients experienced vascular events (4 nonfatal myocardial infarctions, 2 strokes, 4 transient ischemic attacks, 2 intermittent claudications, and 2 had need for percutaneous revascularization procedures). They had higher median values of sCD40L (9.1 versus 5.6 ng/mL; P<0.02) than those who remained event-free.

Of the parameters listed in Table 2, only high sCD40L levels entered the Cox proportional hazard model (P=0.003) (Figure). A separate analysis for hard endpoints (n=6) showed that only sCD40L high values identified patients who experienced critical events (low values=0% versus high values=43%; log-rank test, 17.30; P<0.001).

View this table: [in this window] [in a new window]   TABLE 2. Univariate Analysis

View larger version (11K): [in this window] [in a new window]   Kaplan–Meier curves for ALSC patients grouped by their levels of sCD40L (Cox proportional hazard model, hazard ratio: 5.96; P=0.003). Lines represent subjects with sCD40L above median (solid line, n=14) or below median (broken line, n=28) of patients.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Prospective studies showed that CRP, IL-6, and CD40L predict the risk of CVD,^4,5 and plasma levels of both CRP and IL-6 are independently related to traditional cardiovascular risk factors in women.⁶ Furthermore, sCD40L predicts patients with features of high-risk plaques⁷ and an association between CRP and carotid atherosclerosis has been observed in women.⁸

CD40L may play a pathogenetic role in atherosclerosis. CD40L is rapidly upregulated during platelet activation and triggers an inflammatory response in cells that constitutively express CD40, ie, endothelial cells and monocytes.⁹ CD40 and CD40L are both overexpressed in human and experimental atherosclerotic lesions, particularly in advanced, rupture-prone plaques.¹⁰ Accumulation of T lymphocytes in the fibrous cap may induce macrophages to secrete metalloproteinases via CD40, inducing plaque instability.¹¹ Conversely, CD40L induces tissue factor expression,¹⁰ accounting for thrombotic events within the plaque.

We found increased circulating IL-6, CD40L, and CRP in ALSC and a strong association among these parameters. In the multivariate analysis, only sCD40L above median predict the risk of cardiovascular events, even after adjustment for IL-6, CRP, and CRF. This is consistent with the finding that high CD40L indicates increased risk of coronary events in unstable angina.¹²

Our data confirm that CD40–CD40L interactions represent a main molecular mechanism linking inflammation and thrombosis. Inhibition of this system may represent a potential therapeutic target capable of inducing a more stable carotid plaque phenotype.

	Acknowledgments

 
Supported by grants from the Italian Ministry of Research and Education (40% and Fondo per gli Investimenti della Ricerca di Base) to the "G. d’Annunzio" Foundation, University of Chieti.

Received September 20, 2004; revision received October 29, 2004; accepted November 16, 2004.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. O’Leary DH,Polak JF,Kronmal RA,Manolio TA,Burke GL,Wolfson SK Jr.Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults.N Engl J Med. 1999;340:14–22.[Abstract/Free Full Text]
2. Phipps RP.Atherosclerosis:the emerging role of inflammation and the CD40-CD40L system.Proc Natl Acad Sci U S A. 2000;97:6930–6932.[Free Full Text]
3. Garlichs CD,Kozina S,Fateh-Moghadam S,Handschu R,Tomandl B,Stumpf C,Eskafi S,Raaz D,Schmeisser A,Yilmaz A,Ludwig J,Neundorfer B,Daniel WG.Upregulation of CD40-CD40 ligand (CD154) in patients with acute cerebral ischemia.Stroke. 2003;34:1412–1418.[Abstract/Free Full Text]
4. Ridker PM.Role of inflammatory biomarkers in prediction of coronary heart disease.Lancet. 2001;358:946–948.[CrossRef][Medline] [Order article via Infotrieve]
5. Schönbeck U,Varo N,Libby P,Buring J,Ridker PM.Soluble CD40L and cardiovascular risk in women.Circulation. 2001;104:2266–2268.[Abstract/Free Full Text]
6. Bermudez EA,Rifai N,Buring J,Manson JE,Ridker PM.Interrelationships among circulating interleukin-6, C-reactive protein, and traditional cardiovascular risk factors in women.Arterioscler Thromb Vasc Biol. 2002;22:1668–1673.[Abstract/Free Full Text]
7. Blake GJ,Ostfeld RJ,Yucel EK,Varo N,Schönbeck U,Blake MA,Gerhard M,Ridker PM,Libby P,Lee RT.Soluble CD40 ligand levels indicate lipid accumulation in carotid atheroma. An in vivo study with high-resolution MRI.Arterioscler Thromb Vasc Biol. 2003;23:e11–e14.[Abstract/Free Full Text]
8. Wang TJ,Nam B-H,Wilson PW,Wolf PA,Levy D,Polak JF,D’Agostino RB,O’Donnel CJ.Association of C-reactive protein with carotid atherosclerosis in men and women:the Framingham Heart Study.Arterioscler Thromb Vasc Biol. 2002;22:1662–1667.[Abstract/Free Full Text]
9. Henn V,Slupsky JR,Grafe M,Anagnostoupolos I,Forster R,Muller-Berghaus G,Kroczek RA.CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells.Nature. 1998;391:591–594.[CrossRef][Medline] [Order article via Infotrieve]
10. Lutgens E,Daemen M.CD40-CD40L interactions in atherosclerosis.Trends Cardiovasc Med. 2002;12:27–32.[CrossRef][Medline] [Order article via Infotrieve]
11. Schönbeck U,Mach F,Sukhova GK,Murphy C,Bonnefoy JY,Fabunmi RP,Libby P.Regulation of matrix metalloproteinase expression in human vascular smooth muscle cells by T-lymphocytes. A role for CD40 signaling in plaque rupture?Circ Res. 1997;81:448–454.[Abstract/Free Full Text]
12. Heeschen C,Dimmeler S,Hamm CW,van den Brand MJ,Boersma E,Zeiher AM,Simoons ML,CAPTURE Study Investigators.Soluble CD40 ligand in acute coronary syndromes.N Engl J Med. 2003;348:1104–1111.[Abstract/Free Full Text]

This article has been cited by other articles:

				C. T. Damsgaard, H. Frokiaer, A. D. Andersen, and L. Lauritzen Fish Oil in Combination with High or Low Intakes of Linoleic Acid Lowers Plasma Triacylglycerols but Does Not Affect Other Cardiovascular Risk Markers in Healthy Men J. Nutr., June 1, 2008; 138(6): 1061 - 1066. [Abstract] [Full Text] [PDF]

				R. R. S. Packard and P. Libby Inflammation in Atherosclerosis: From Vascular Biology to Biomarker Discovery and Risk Prediction Clin. Chem., January 1, 2008; 54(1): 24 - 38. [Abstract] [Full Text] [PDF]

				D. Ferro, L. Loffredo, L. Polimeni, F. Fimognari, P. Villari, P. Pignatelli, V. Fuster, and F. Violi Soluble CD40 Ligand Predicts Ischemic Stroke and Myocardial Infarction in Patients With Nonvalvular Atrial Fibrillation Arterioscler. Thromb. Vasc. Biol., December 1, 2007; 27(12): 2763 - 2768. [Abstract] [Full Text] [PDF]

				K. Minoguchi, T. Yokoe, T. Tazaki, H. Minoguchi, N. Oda, A. Tanaka, M. Yamamoto, S. Ohta, C. P. O'Donnell, and M. Adachi Silent Brain Infarction and Platelet Activation in Obstructive Sleep Apnea Am. J. Respir. Crit. Care Med., March 15, 2007; 175(6): 612 - 617. [Abstract] [Full Text] [PDF]

				E. Corrado, M. Rizzo, R. Tantillo, I. Muratori, F. Bonura, G. Vitale, and S. Novo Markers of Inflammation and Infection Influence the Outcome of Patients With Baseline Asymptomatic Carotid Lesions: A 5-Year Follow-Up Study Stroke, February 1, 2006; 37(2): 482 - 486. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 36/3/673    most recent 01.STR.0000154878.58398.14v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Novo, S. Articles by Davì, G. Search for Related Content PubMed PubMed Citation Articles by Novo, S. Articles by Davì, G. Pubmed/NCBI databases Substance via MeSH Medline Plus Health Information Stroke Related Collections Risk Factors Carotid Stenosis Doppler ultrasound, Transcranial Doppler etc.