Original Paper Received: May 6, 2014 Accepted: July 8, 2014 Published online: October 9, 2014

Cerebrovasc Dis 2014;38:204–211 DOI: 10.1159/000365841

Plasma CX3CL1 Levels and Long Term Outcomes of Patients with Atrial Fibrillation: The West Birmingham Atrial Fibrillation Project Yutao Guo a, b Stavros Apostalakis a Andrew D. Blann a Gregory Y.H. Lip a  

a

 

 

 

Haemostasis, Thrombosis and Vascular Biology Unit, University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, UK; b Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing, China  

 

Key Words Atrial fibrillation · Stroke · Thromboembolism · Chemokines · CX3CL1

Abstract Background: There is growing evidence that chemokines are potentially important mediators of the pathogenesis of atherosclerotic disease. Major atherothrombotic complications, such as stroke and myocardial infarction, are common among atrial fibrillation (AF) patients. This increase in risk of adverse events may be predicted by a score based on the presence of certain clinical features of chronic heart failure, hypertension, age 75 years or greater, diabetes and stroke (the CHADS2 score). Our objective was to assess the prognostic value of plasma chemokines CCL2, CXCL4 and CX3CL1, and their relationship with the CHADS2 score, in AF patients. Methods: Plasma CCL2, CXCL4 and CX3CL1 were measured in 441 patients (59% male, mean age 75 years, 12% paroxysmal, 99% on warfarin) with AF. Baseline clinical and demographic factors were used to define each subject’s CHADS2 score. Patients were followed up for a mean 2.1 years, and major adverse cardiovascular and cerebrovascular events (MACCE) were sought, being the combination of cardiovascular death, acute coronary events, stroke and systemic embolism. Results: Fifty-five of the AF patients suffered a MACCE (6% per year). Those in the lowest CX3CL1 quartile (≤0.24 ng/ml) had fewest MACCE (p = 0.02). In the Cox regression analysis, CX3CL1 levels >0.24 ng/ml (Hazard ratio

© 2014 S. Karger AG, Basel 1015–9770/14/0383–0204$39.50/0 E-Mail [email protected] www.karger.com/ced

2.8, 95% CI 1.02–8.2, p = 0.045) and age (p = 0.042) were independently linked with adverse outcomes. The CX3CL1 levels rose directly with the CHADS2 risk score (p = 0.009). The addition of CX3CL1 did not significantly increased the discriminatory ability of the CHADS2 clinical factor-based risk stratification (c-index 0.60 for CHADS2 alone versus 0.67 for CHADS2 plus CX3CL1 >0.24 ng/ml, p = 0.1). Aspirin use was associated with lower levels of CX3CL1 (p = 0.0002) and diabetes with higher levels (p = 0.031). There was no association between CXCL4 and CCL2 plasma levels and outcomes. Conclusion: There is an independent association between low plasma CX3CL1 levels and low risk of major cardiovascular events in AF patients, as well as a linear association between CX3CL1 plasma levels and CHADS2-defined cardiovascular risk. The potential for CX3CL1 in refining risk stratification in AF patients merits consideration. © 2014 S. Karger AG, Basel

Introduction

Inflammation, platelet activation and atrial fibrillation (AF) are closely linked [1–3]. Inflammatory mediators such as C-reactive protein (CRP), and cytokines such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-6, and IL-8 have been associated with both the pres-

Yutao Guo and Stavros Apostolakis contributed equally to this work.

Dr. Andrew D. Blann University of Birmingham Centre for Cardiovascular Sciences City Hospital, Dudley Road Birmingham, B18 7QH (UK) E-Mail a.blann @ bham.ac.uk

ence and the outcomes of AF, such as stroke [4–7]. Cytokines and chemokines may have a functional role in thrombosis associated with AF by promoting platelet activation and increasing expression and release of coagulation factors and other procoagulants [7–11]. CCL2 (also known as monocyte chemoattractive protein), a chemokine family member, has been considered a potent monocyte chemotactic cytokine with a potentially important role in atherosclerosis. For example, CCL2 levels are elevated during episodes of AF [12–14]. CX3CL1 (also known as fractalkine), the sole member of the CX3C chemokine family, has also been shown to play a unique role in atherosclerosis, acting both as a chemoattractant and as an adhesion molecule for monocytes, and a mediator of thrombosis through platelet activation [15, 16]. CXCL4 (also known as platelet factor 4) is a member of the CXC family that was originally considered a platelet activator, although recent evidence proposes a more robust role in inflammatory pathways leading to atherothrombosis [17–19]. Increased CXCL4 levels have been found in patients with AF [20], while CX3CL1 was found to be an independent predictor of mortality in patients with advanced heart failure [21]. Platelets are rich sources of chemokine ligands and express chemokine receptors. Inflammation is a major mediator of platelet activation and thus a potential contributor of the prothrombotic state associated with AF [3, 11, 22–27]. CXCL4 and CCL2 are found in platelet α-granules, while CX3CL1 receptor (CX3CL1R1) is expressed on human platelets [28]. In a flow chamber model of platelet adhesion, stimulation with CX3CL1 significantly enhanced platelet adhesion to collagen and fibrinogen [29]. CX3CL1 is an unusual chemotactic factor existing in both membrane-bound and soluble form, acting as an adhesion molecule and chemoattractant respectively [16, 30]. Several lines of evidence support a role for CX3CL1 in atherosclerotic cardiovascular disease [31–33]: it has potent pro-atherosclerotic effects including the promotion of proliferation, migration and activation of leukocytes in the vessel wall, cytotoxic effects on the endothelium, antiapoptotic and proliferative effects on human vascular smooth muscle cells and platelet activation. We hypothesised that plasma chemokines CCL2, CX3CL1 and CXCL4 have a role in AF, raising the possibility that plasma levels of the molecules could be used to identify patients with a high risk of atherothrombotic complications. We tested this hypothesis in two experiments – first, by determining the relationship between the cytokines and the CHADS2 formula for risk of stroke [34], and second, by conducting a two-year follow-up CX3CL1 Levels and Long Term Outcomes in AF

study to determine the extent to which any of the chemokines could predict a composite of major cardiovascular end points. Materials and Methods Patients and Design Patients with AF were prospectively recruited as part of the West Birmingham AF project during their visit to the outpatient cardiology clinic, and the anticoagulation clinics in Sandwell and West Birmingham Hospitals. Patients were enrolled if they were ≥18 years old and if they had a documented episode of AF by an ECG or 24 h ambulatory heart rate recording. Patients were excluded from participation if they were aged 0.24 ng/ml) in a binary regression model along with the factors that comprise the CHADS2 score, it increased the discriminatory performance of the model for MACCEs by over 10% (c-index 0.67, 95% CI 0.58–0.75 vs. 0.60, 95% CI 0.51–0.70). However, this improvement failed to reach statistical significance (p = 0.1) (fig. 3). No 208

Cerebrovasc Dis 2014;38:204–211 DOI: 10.1159/000365841

statistically significant association was established between CCL2 or CXCL4 plasma levels and outcomes or thromboembolic risk profile of patients with AF.

Discussion

In a prospectively recruited cohort of AF patients, we determined the value of three plasma chemokine levels (all possibly linked to the pathogenesis of cardiovascular disease [37]) as prognostic markers of major adverse cardiovascular and cerebrovascular events (MACCE). High CX3CL1 plasma levels were associated with a poor outcome, a finding that remained significant after multivariate adjustment for demographic and clinical factors. We also observed a linear association between plasma CX3CL1 and thromboembolic risk profile, as defined by the CHADS2 score. Although in a population with a different degree of atherosclerosis and anti-thrombotic therapy, we confirm the data of Njerve et al. [38], who reported no effect of diabetes, but that smoking reduced levels of CX3CL1. In this study, CX3CL1 independently predicted the risk for MACCE. This result was mostly driven by the higher risk for cardiovascular mortality. In agreement with these results, Richter et al. demonstrated that CX3CL1 is an independent predictor of cardiovascular mortality in heart failure [21]. A major difference between this study and ours is that the present study demonstrated a predominantly protective effect of low CX3CL1 levels rather than increased risk with high CX3CL1 levels. We are unable to confirm a statistically significant association between CX3CL1 and age or eGFR [21]. Furthermore, with the exception of smoking, we failed to demonstrate convincing interactions between plasma CX3CL1 levels and individual cardiovascular co-morbidities or risk factors. Nevertheless, we observed a clear linear association between CX3CL1 levels and thromboembolic risk as reflected by the CHADS2 score. Notably, Donohue et al. [39] found that low levels of CX3CL1 were a risk factor for MACCE, but in those who had already suffered a stroke, and so would be more likely to be treated with aspirin. We found the reverse, where over 95% of our patients were not taking aspirin. Indeed, 18 of our 20 patients taking aspirin had levels of CX3CL1 lower than the median of the entire group, leading us to conclude that this drug reduces the levels of the chemokine. We selected the CHADS2 score as a clinical marker of cardiovascular outcome since it is a well-characterised risk estimation score in AF patients [34]. High CHADS2 Guo/Apostalakis/Blann/Lip

Table 5. Event rates in the cohort stratified by quartiles of chemokine

N (%)

All MACCE Cardiovascular mortality All-cause mortality Acute coronary syndromes

All MACCE Cardiovascular mortality All-cause mortality Acute coronary syndromes

All MACCE Cardiovascular mortality All-cause mortality Acute coronary syndromes

Quartiles of CX3CL1, ng/ml ≤0.24

0.25–0.47

0.48–0.90

≥0.91

40 (100.0) 21 (52.5) 29 (72.5) 20 (50.0)

4 (10.0) 1 (4.8) 4 (13.8) 3 (15.0)

13 (32.5) 6 (28.6) 9 (31.0) 8 (40.0)

15 (37.5) 9 (42.8) 11 (37.9) 6 (30.0)

8 (20.0) 5 (23.8) 5 (17.2) 3 (15.0)

N (%)

Quartiles of CCL2, pg/ml ≤27

28–54

55–110

≥111

38 (100) 18 (47.4) 27 (71.0) 20 (52.6)

7 (18.4) 3 (16.7) 5 (18.5) 4 (20.0)

9 (23.7) 5 (27.8) 7 (25.9) 4 (20.0)

14 (36.8) 8 (44.4) 12 (44.4) 6 (30.0)

8 (21.0) 2 (11.1) 3 (11.1) 6 (30.0)

N (%)

Quartiles of CXCL4, ng/ml

44 (10.00) 21 (47.7) 32 (72.7) 22 (50.0)

≤1.08

1.09–3.56

3.57–10.1

≥10.2

10 (22.7) 4 (19.0) 6 (18.7) 8 (36.4)

11 (25.0) 9 (42.8) 9 (28.1) 3 (13.6)

8 (18.2) 11 (52.4) 7 (21.9) 5 (22.7)

15 (34.1) 5 (23.8) 10 (31.2) 6 (27.3)

P1

P2

0.04 0.09 0.19 0.29

0.02 0.03 0.18 0.43

P1

P2

0.33 0.18 0.06 0.84

0.43 0.58 0.50 0.79

P1

P2

0.43 0.19 0.72 0.49

0.86 0.13 0.53 0.21

Color version available online

P1 refers to the overall chi square analysis. P2 refers to comparison of the lower quartile with the remaining quartiles. MACCE (major adverse cardiovascular and cerebrovascular events) represents the combined endpoint of cardiovascular mortality, all-cause mortality, acute coronary syndromes and stroke/TIA.

1.00

Fig. 2. Cumulative major adverse cardiovas-

cular and cerebrovascular events (MACCE) free survival of patients with CX3CL1 levels ≤0.24 ml (lower quartile) and patients with CX3CL1 >0.24 ng/ml. The model included age, gender, history of stroke/TIA, hypertension, diabetes and congestive heart failure as co-variates. MACCE includes: stroke/TIA, acute coronary events and cardiovascular death.

CX3CL1 Levels and Long Term Outcomes in AF

Cumulative survival

0.95 p = 0.042 0.90

0.85 CX3CL1 >0.24 ng/ml CX3CL1 >0.24 ng/ml

0.80 0

200

400

600

800

1,000

Time of first MACCE (days)

Cerebrovasc Dis 2014;38:204–211 DOI: 10.1159/000365841

209

Color version available online

1.0

Sensitivity

0.8

0.6

0.4

0.2 CHADS2 0

CHADS2 plus CX3CL1 0

0.2

0.4

0.6

0.8

1.0

1 – specificity

tion of clinically relevant associations between each chemokine and individual endpoints. Moreover, chemokine levels were measured once during follow-up and it is likely that high chemokine levels in a certain time point might be the result of a transient inflammatory condition and not the outcome of a chronic vascular disease. Having failed to measure an established inflammatory marker (CRP, IL-6), we cannot comment further on this point. A further limitation is that when the study was designed and undertaken, we used CHADS2, and so we are unable to consider our data in terms of the recently developed CHA2DS2VASc score. In conclusion, we report an independent association between low CX3CL1 plasma levels and low rates of the combined endpoint of stroke/TIA, acute coronary syndrome and cardiovascular death in patients with AF, as well as a linear association between CX3CL1 plasma levels  and estimated thromboembolic risk. The impact of CX3CL1 in refining risk stratification in AF patients merits consideration.

Fig. 3. Receiver operator characteristic analysis of CHADS2 score

and CHADS2 plus CX3CL1 >0.24 ng/ml for the outcome of major adverse cardiovascular and cerebrovascular events (MACCE). Cindex 0.60, 95% CI 0.51–0.7 vs. 0.67, 95% CI 0.58–0.75 respectively, Z = 1.6, p = 0.1.

score in AF populations is not associated merely with increased risk of stroke/thromboembolism but also with higher risk of adverse cardiovascular outcomes including  cardiovascular mortality [1, 40]. The association of CX3CL1 levels and CHADS2 score instead of with individual comorbidities is likely a reflection of the multi-factorial background of AF. We note several limitations. Our study population was almost entirely anticoagulated patients with a relatively good TTR, and so the occurrence of thromboembolism was low. Accordingly, it is underpowered for the detec-

Addendum All authors contributed to the concept and design and were involved in the interpretation of data, critical writing and revising the intellectual content, and gave final approval to the version to be published. Drs. Guo, Apostolakis and Blann performed the laboratory work, the statistical analysis, and provided the first draft of the manuscript.

Acknowledgements We gratefully thank Drs. Tay Kok Hoon and Deirdre Lane for collecting the samples and completing the baseline data, and Balu Balakrishnan for laboratory assistance. The study was financially supported by University of Birmingham research funds and a European Society of Cardiology 2010–2011 Atherothrombosis Research Grant.

References 1 Lip GY, Tse HF, Lane DA: Atrial fibrillation. Lancet 2012;379:648–661. 2 Camm AJ, Lip GY, De Caterina R, Savelieva I, Atar D, Hohnloser SH, Hindricks G, Kirchhof P; ESC Committee for Practice GuidelinesCPG: An update of the 2010 ESC Guidelines for the management of atrial fibrillation. Europace 2012;14:1385–1413. 3 Guo Y, Lip GY, Apostolakis S: Inflammation in atrial fibrillation. J Am Coll Cardiol 2012; 60:2263–2270.

210

4 Schnabel RB, Larson MG, Yamamoto JF, Kathiresan S, Rong J, Levy D, Keaney JF Jr, Wang TJ, Vasan RS, Benjamin EJ: Relation of multiple inflammatory biomarkers to incident atrial fibrillation. Am J Cardiol 2009;104:92–96. 5 Ederhy S, Di Angelantonio E, Dufaitre G, Meuleman C, Masliah J, Boyer-Chatenet L, Boccara F, Cohen A: C-reactive protein and transesophageal echocardiographic markers of thromboembolism in patients with atrial fibrillation. Int J Cardiol 2012;159:40–46.

Cerebrovasc Dis 2014;38:204–211 DOI: 10.1159/000365841

6 Hak Ł, Myśliwska J, Wieckiewicz J, Szyndler K, Siebert J, Rogowski J: Interleukin-2 as a predictor of early postoperative atrial fibrillation after cardiopulmonary bypass graft (CABG). J Interferon Cytokine Res 2009;29:327–332. 7 Pinto A, Tuttolomondo A, Casuccio A, Di Raimondo D, Di Sciacca R, Arnao V, Licata G: Immuno-inflammatory predictors of stroke at follow-up in patients with chronic non-valvular atrial fibrillation (NVAF). Clin Sci (Lond) 2009;116:781–789.

Guo/Apostalakis/Blann/Lip

8 Li-Saw-Hee FL, Blann AD, Lip GY: A crosssectional and diurnal study of thrombogenesis among patients with chronic atrial fibrillation. J Am Coll Cardiol 2000;35:1926–1931. 9 Cardona SM, Garcia JA, Cardona AE: The fine balance of chemokines during disease: trafficking, inflammation and homeostasis. Methods Mol Biol 2013;1013:1–16. 10 Marín F, Corral J, Roldán V, González-Conejero R, del Rey ML, Sogorb F, Lip GY, Vicente V: XIII Val34Leu polymorphism modulates the prothrombotic and inflammatory state associated with atrial fibrillation. J Mol Cell Cardiol 2004;37:699–704. 11 Akar JG, Jeske W, Wilber DJ: Acute onset human atrial fibrillation is associated with local cardiac platelet activation and endothelial dysfunction. J Am Coll Cardiol 2008; 51: 1790–1793. 12 Arakelyan A, Petrkova J, Hermanova Z, Boyajyan A, Lukl J, Petrek M: Plasma levels of the CCL2 chemokine in patients with ischemic stroke and myocardial infarction. Mediat Inflamm 2005;3:175–179. 13 Deo R, Khera A, McGuire DK, Murphy SA, Meo Neto Jde P, Morrow DA, de Lemos JA: Association among plasma levels of monocyte chemoattractant protein-1, traditional cardiovascular risk factors, and subclinical atherosclerosis. J Am Coll Cardiol 2004; 44: 1812–1818. 14 Hammwöhner M, Ittenson A, Dierkes J, Bukowska A, Klein HU, Lendeckel U, Goette A: Platelet expression of CD40/CD40 ligand and its relation to inflammatory markers and adhesion molecules in patients with atrial fibrillation. Exp Biol Med (Maywood) 2007; 232: 581–589. 15 Meyer dos Santos S, Klinkhardt U, Scholich K, Nelson K, Monsefi N, Deckmyn H, Kuczka K, Zorn A, Harder S: The CX3C chemokine CX3CL1 mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib. Blood 2011;117:4999–5008. 16 Liu H, Jiang D: Fractalkine/CX3CR1 and atherosclerosis. Clin Chim Acta 2011;412:1180– 1186. 17 Hirsh J: Blood tests for the diagnosis of venous and arterial thrombosis. Blood 1981;57: 1–8. 18 Miller MD, Krangel MS: Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines. Crit Rev Immunol 1992;12:17–46.

CX3CL1 Levels and Long Term Outcomes in AF

19 Kasper B, Petersen F: Molecular pathways of platelet factor 4/CXCL4 signaling. Eur J Cell Biol 2011;90:521–526. 20 Ohara K, Inoue H, Nozawa T, Hirai T, Iwasa A, Okumura K, Lee JD, Shimizu A, Hayano M, Yano K: Accumulation of risk factors enhances the prothrombotic state in atrial fibrillation. Int J Cardiol 2008;126:316–321. 21 Richter B, Koller L, Hohensinner PJ, Rychli K, Zorn G, Goliasch G, et al: Fractalkine is an independent predictor of mortality in patients with advanced heart failure. Thromb Haemost 2012;108:1220–1227. 22 Roldán V, Marín F, Blann AD, García A, Marco P, Sogorb F, Lip GY: Interleukin-6, endothelial activation and thrombogenesis in chronic atrial fibrillation. Eur Heart J 2003;24: 1373–1380. 23 Conway DS, Buggins P, Hughes E, Lip GY: Relation of interleukin-6, C-reactive protein, and the prothrombotic state to transesophageal echocardiographic findings in atrial fibrillation. Am J Cardiol 2004;93:1368–1373. 24 Conway DS, Buggins P, Hughes E, Lip GY: Relationship of interleukin-6 and C-reactive protein to the prothrombotic state in chronic atrial fibrillation. J Am Coll Cardiol 2004; 43: 2075–2082. 25 Watson T, Shantsila E, Lip GY: Mechanisms of thrombogenesis in atrial fibrillation: Virchow’s triad revisited. Lancet 2009; 373: 155– 166. 26 Hayashi M, Takeshita K, Inden Y, Ishii H, Cheng XW, Yamamoto K, Murohara T: Platelet activation and induction of tissue factor in acute and chronic atrial fibrillation: involvement of mononuclear cell-platelet interaction. Thromb Res 2011;128:e113–e118. 27 Herrera Siklódy C, Arentz T, Minners J, Jesel L, Stratz C, Valina CM, Weber R, Kalusche D, Toti F, Morel O, Trenk D: Cellular damage, platelet activation, and inflammatory response after pulmonary vein isolation: a randomized study comparing radiofrequency ablation with cryoablation. Heart Rhythm 2012;9:189–196. 28 Gleissner CA, von Hundelshausen P, Ley K: Platelet chemokines in vascular disease. Arterioscler Thromb Vasc Biol 2008; 28: 1920– 1927. 29 Schäfer A, Schulz C, Eigenthaler M, Fraccarollo D, Kobsar A, Gawaz M, Ertl G, Walter U, Bauersachs J: Novel role of the membranebound chemokine CX3CL1 in platelet activation and adhesion. Blood 2004;103:407–412. 30 Bazan JF, Bacon KB, Hardiman GJ: A new class of membrane bound chemokine with a CX3C motif. Nature 1997;385:640–644.

31 Li J, Guo Y, Luan X, Qi T, Li D, Chen Y, Ji X, Zhang Y, Chen W: Independent roles of monocyte chemoattractant protein-1, regulated on activation, normal T-cell expressed and secreted and fractalkine in the vulnerability of coronary atherosclerotic plaques. Circ J 2012;76:2167–2173. 32 Flierl U, Schäfer A: Fractalkine – a local inflammatory marker aggravating platelet activation at the vulnerable plaque. Thromb Haemost 2012;108:457–463. 33 Husberg C, Nygård S, Finsen AV, Damås JK, Frigessi A, Oie E, Waehre A, Gullestad L, Aukrust P, Yndestad A, Christensen G: Cytokine expression profiling of the myocardium reveals a role for CX3CL1 (fractalkine) in heart failure. J Mol Cell Cardiol 2008; 4: 261– 269. 34 Gage BF, Waterman AD, Shannon W, Boechler M, Rich MW, Radford MJ: Validation of clinical classification schemes for predicting stroke: results from the National Registry of Atrial Fibrillation. JAMA 2001; 285: 2864–2870. 35 DeLong ER, DeLong DM, Clarke-Pearson DL: Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics 1988;44:837–845. 36 Altman DG: Practical statistics for medical research. Chapman and Hall, London, 1991. 37 Zernecke A, Shagdarsuren E, Weber C: Chemokines in atherosclerosis: an update. Arterioscler Thromb Vasc Biol 2008; 28: 1897– 1908. 38 Njerve IU, Pettersen AÅ, Opstad TB, Arnesen H, Seljeflot I: Fractalkine and its receptor (CX3CR1) in patients with stable coronary artery disease and diabetes mellitus. Metab Syndr Relat Disord 2012; 10: 400– 406. 39 Donohue MM, Cain K, Zierath D, Shibata D, Tanzi PM, Becker KJ: Higher plasma fractalkine is associated with better 6-month outcome from ischemic stroke. Stroke 2012; 43: 2300–2306. 40 Oldgren J, Alings M, Darius H, Diener HC, Eikelboom J, Ezekowitz MD, et al: Risks for stroke, bleeding, and death in patients with atrial fibrillation receiving dabigatran or warfarin in relation to the CHADS2 score: a subgroup analysis of the RE-LY trial. Ann Intern Med 2011;155:660–667.

Cerebrovasc Dis 2014;38:204–211 DOI: 10.1159/000365841

211

Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.

Plasma CX3CL1 levels and long term outcomes of patients with atrial fibrillation: the West Birmingham Atrial Fibrillation Project.

There is growing evidence that chemokines are potentially important mediators of the pathogenesis of atherosclerotic disease. Major atherothrombotic c...
215KB Sizes 0 Downloads 7 Views