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Increased plasma C-reactive protein and interleukin-6 concentrations in patients with slow coronary flow
Clinica Chimica Acta 385 (2007) 43 – 47 www.elsevier.com/locate/clinchim
Increased plasma C-reactive protein and interleukin-6 concentrations in patients with slow coronary flow Jian-Jun Li ⁎, Xue-Wen Qin, Zi-Cheng Li 1 , He-Song Zeng 2 , Zhan Gao, Bo Xu, Chao-Yang Zhang, Jie Li Department of Cardiology, Fu Wai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100037, People's Republic of China Received 16 June 2006; received in revised form 30 May 2007; accepted 31 May 2007 Available online 15 June 2007
Abstract Background: Slow coronary filling of epicardial coronary arteries in the absence of stenosis is not infrequently detected finding during routine coronary angiography. There is mounting evidence suggested that an inflammatory process play an important role in atherosclerotic pathogenesis appeared in different clinical settings. However, the possible association between inflammation and slow coronary flow (SCF) has not been investigated. We examined whether the increased inflammatory markers are present in patients with SCF. Methods: Forty-two patients with SCF detected by coronary angiography via the Thrombosis In Myocardial Infarction (TIMI) frame count method were enrolled in this study. The plasma concentration of high-sensitivity C-reactive protein (CRP) and interleukin-6 (IL-6) were evaluated using commercial available kits. Data were compared with 30 control subjects with angiographically normal coronary flow. Results: There are no differences regarding clinical characteristics between the 2 groups. The data showed, however, that plasma CRP and IL-6 concentrations were higher in patients with SCF compared with normal control subject (CRP: 0.27 ± 0.16 vs. 0.22 ± 0.11mg/l; and IL-6: 8.7 ± 0.8 vs. 5.4 ± 0.4pg/ml, p b 0.01 respectively). In addition, mean TIMI frame count was positively correlated with plasma CRP and IL-6 concentrations (CRP: γ = 0.551; IL-6: γ = 0.573, p b 0.01 respectively). Conclusions: Plasma concentration of CRP and IL-6 concentrations increased, and was positive correlated with TIMI frame count in patients with SCF compared with normal coronary flow subject. Therefore, whether the increased inflammatory markers are related to the pathogenesis of SCF in these patients deserved further investigation. © 2007 Elsevier B.V. All rights reserved. Keywords: Inflammation, C-reactive protein, Interleukin-6, Slow coronary flow
1. Introduction The slow coronary flow (SCF) phenomenon is an angiographic observation characterized by angiographically normal or near-normal coronary arteries with delayed opacification of the distal vasculature [1–3]. It has been reported that coronary endothelial dysfunction play an important pathogenetic role in patients with SCF. Moreover, myocardial biopsy studies have also revealed the presence of coronary microvascular disease in ⁎ Corresponding author. Tel.: +86 10 88396077; fax: +86 10 68331730. E-mail address: [email protected] (J.-J. Li). 1 Division of Cardiology, First Affiliated Hospital, Jinan University, Guangzhou 510630, PR China. 2 Cardiovascular Division, Tongji Hospital of Tongji Medical College, Huazhong University, of Science and Technology, Wuhan 430030, PR China. 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.05.024
patients exhibiting SCF [4]. However, the precise mechanisms responsible for this microvascular endothelial dysfunction in patients with SCF are still unknown. Although atherosclerosis has been considered to be multifactorial disease in which genetic, environmental, metabolic factors have been implicated, the gaps remain in our knowledge of the etiopathogenesis of atherosclerosis. There is mounting evidence that inflammation plays an important role in the initiation, development as well as evolution of atherosclerosis, suggesting that atherosclerosis is an inflammation disease [5–8]. Although triggers and pathways of inflammation are probably multiple and different in different clinical settings, the data from animals as well as humans including our groups indicated that an inflammatory process was involved in all stages of atherosclerosis appeared in different clinical entities [9–11].
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J.-J. Li et al. / Clinica Chimica Acta 385 (2007) 43–47
In addition, a large amount of data indicated that C-reactive protein (CRP) is a sensitive marker of underlying systemic inflammation, are increased among men and women at risk for future cardiovascular events, and the addition of CRP testing to standard lipid screening seems to provide an improved method to determine vascular risk [6]. These data, as well as accumulating evidence that CRP may have direct inflammatory effects at the endothelial levels. Besides, among pro-inflammatory cytokines, interleukin-6 (IL-6) is one of the most important factors and has multiple important effects in human pathphysiology. In our previous study, the data showed that an enhanced production of IL-6 by monocytes in response to CRP in patients with unstable angina [10]. Moreover, IL-6 is a powerful stimulus for CRP production [11]. Based on those observations, we hypothesis that an inflammatory process may be associated with the development of SCF [9]. 2. Methods 2.1. Subjects The protocol of the study were approved by the Ethics Review Board of Fu Wai Hospital, Chinese Academy of Medical Science and Peking Union Medical College, and Tongji Hospital of Tongji Medical College, Huazahong University of Science and Technology, and First Affiliated Hospital of Jinan University, and all the patients provided informed consent. The study groups included 42 consecutive patients referred for assessment to our centers for Diagnosis and Treatment of Coronary Artery Disease. Entry criteria were patients with angiographically proven normal coronary arteries and show SCF in all 3 coronary vessels (SCF group, 34 males and 8 females, mean age 49 ± 7y), and 30 subjects with angiographically proven normal coronary artery with normal coronary flow (NCF group, 25 males and 5 females, mean age 50 ± 8y). All patients with SCF were selected from individuals who underwent coronary angiography in our centers with a suspicion of coronary artery disease and diagnosed as having angiographically normal coronary arteries. Control subjects consisted of 30 consecutive subjects with atypical chest pain admitted to our center or division for selective coronary angiography and subsequently found to have angiographically normal coronary arteries with normal coronary flow. All subjects enrolled in this study had normal hepatic and renal function. The hyperlipidemia was defined as low-density lipoprotein cholesterol ≥ 160mg/dl or total cholesterol ≥ 220mg/dl and/or triglyceride (TG) ≥ 200mg/dl. Patients with evidence of coronary artery disease, myocardial infarction, valvular heart disease, congestive heart failure, left ventricular dysfunction, echocardiographically proven left ventricular hypertrophy, a history of dysphagia, swallowing as well as intestinal motility disorders, untreated thyroid disease, sinus node dysfunction or conduction disturbance, estrogen replacement therapy, carcinoma, poorly controlled hypertension (systolic blood pressure N 160mm Hg or diastolic blood pressure N 105mm Hg), recent major operation (b 3months), autoimmune disease, metabolic syndrome and infection were excluded from the study.
2.2. Coronary angiography Left ventricular and selective coronary angiography for all enrolled patients was performed using the standard Judkin's techniques, and the results were analyzed by at least 2 interventional physicians according to our previous study. Only angiograms with visually smooth contours with no wall irregularities were considered as normal. Contrast used in angiography of this study is Iopromide (Ultravist-370, Schering AG, Berlin, Germany). Coronary flow rates of all subjects were determined by Thrombosis In Myocardial Infarction frame count (TIMI frame count) because the method is a simple, reproducible, objective and quantitative index of coronary flow velocity [12,13]. TIMI frame count was determined for each major coronary artery in each patient and control subject according to the method first described by Gibson et al. [12]. In brief, the number of cineangiographic frames, recorded at
30fps, required for the leading edge of the column of radiographic contrast to reach a predetermined landmark, is determined. The first frame is defined as the frame in which concentrated dye occupies the full width of the proximal coronary artery lumen, touching both borders of the lumen, and forward motion down the artery. The final frame is designated when the leading edge of the contrast column initially arrives at the distal landmark. In the left anterior descending (LAD) coronary artery, the landmark used is the most distal branch nearest the apex of the left ventricle, commonly referred as the “pitchfork”. All anti-anginal and anti-ischemic medications, except sublingual nitroglycerin, were withheld for at least 24h before the examination. During coronary angiography, to exclude the possibility of coronary spasm, all patients underwent hyperventilation tests, which were performed by asking the patients to breathe quickly and deeply for at least 5min.
2.3. CRP and IL-6 determinations EDTA-anticoagulated peripheral blood sample were taken after 12-h overnight fast at baseline (before coronary angiography). The plasma was obtained after a centrifugation of 3000rpm at 4°C for 15min. The concentrations of high-sensitivity CRP were determined using immunoturbidometry (Beckmann Assay 360) as our previously reported [10]. The median normal value for CRP is 0.8mg/l, with 90% of normal values b 0.3mg/l, with a lower detection limit of 0.2mg/l. The inter-assay CVs were 4.4% and 4.8%, respectively, and intra-assay CVs were 3.5% and 5.1%, respectively. IL-6 was measured with a commercial assay kit (Quantikine human IL-6, R & D System). IL-6 measurements were performed from plasma in duplicate, and both intra- and inter-assay CV was b 10%. The range of values detected by the assay for IL-6 was 3 to 5000pg/ml as our previously reported [11].
2.4. Statistical analysis Continuous variables are expressed as mean ± SD, and categorical variables were expressed as percentage. Comparison of categorical and continuous variables between the two groups was performed using chi-square test and unpaired t test, respectively. Because the distribution of CRP is skewed rightward, log transformation was made at baseline, and the significance of any difference in distributions was assessed by the Wilcoxon rank–sum test as our previously reported [11]. The correlation among each major epicardial coronary arteries and between the plasma concentrations of log-CRP or IL-6 and mean TIMI frame count was assessed by the Pearson correlation test. A p b 0.05 was considered statistically significant.
Table 1 Baseline clinical characteristics (mean ± SD) Variables
SFC group
NCF group
p value
(n = 42)
(n = 30)
Age (y) Male/female Body mass index (kg/m2) Family history of CAD, n (%) Current smoker, n (%) Hypertension, n (%) Hperlipidemia, n (%) Diabetes, n (%) EF (%)
49 ± 7 34/8 23 ± 4 5 (12) 10 (24) 11 (26) 5 (12) 2 (5) 60 ± 8
50 ± 8 25/5 22 ± 4 2 (7) 6 (20) 5 (17) 4 (13) 1 (3) 59 ± 7
NS NS NS NS NS NS NS NS NS
Medications Aspirin, n (%) β-blocker, n (%) ACEI, n (%) Statin, n (%) CCB, n (%)
42 (100) 27 (64) 10 (24) 5 (12) 4 (10)
30 (100) 21 (70) 3 (10) 4 (13) 2 (7)
NS NS NS NS NS
CAD = coronary artery disease; NS = not significance; ACEI = angiotensinconverting enzyme inhibitor; CCB = calcium channel blocker; EF = ejection fraction.
J.-J. Li et al. / Clinica Chimica Acta 385 (2007) 43–47
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3. Results 3.1. No difference of baseline clinical characteristics between the two groups Baseline clinical characteristics of subjects with slow coronary flow (n = 42) and with angiographically normal coronary flow (n = 30) are given in Table 1. There are no differences regarding clinical characteristics between the 2 groups including age, sex, body mass index, history of coronary artery disease, smoker, hypertension, hyperlipidemia, diabetes, and medications used before. 3.2. TIMI frame count and vessels diameter TIMI frame counts including LAD, left circumflex artery (LCX), right coronary artery (RCA) and vessels diameters are presented in Table 2. The data showed that patients with SCF were detected to have significantly higher TIMI frame count compared to normal control subjects for each major epicardial coronary artery. Mean TIMI frame count was found to be significantly higher in patients with SCF compared with normal control subjects (47.6 ± 16.2 vs 28.2 ± 3.0, p b 0.01). However, mean epicardial coronary artery diameters were detected to be similar in patients with SCF and normal control subjects (3.03 ± 0.20 vs. 3.01 ± 0.21mm, p N 0.05).
Fig. 1. Comparison of mean CRP (A) and IL-6 (B) between slow coronary flow group (SCF group, n = 42) and normal coronary flow group (NCF group, n = 30). Data are mean ± S.D. ⁎p b 0.01 compared with NCF group. CRP = C-reactive protein; IL-6 = interleukin-6.
3.3. Increased CRP and IL-6 concentrations in patients with SCF
3.4. Correlation among each major epicardial coronary arteries
As showed in Table 2 and Fig. 1A and B, the plasma CRP concentrations were higher in patients with SCF than that in subjects with normal coronary flow (0.27 ± 0.16 vs. 0.22 ± 0.11mg/l, p b 0.01). In addition, the pattern of plasma IL-6 concentrations was also similar as CRP concentrations. That is, the increased plasma IL-6 concentration was also found in patients with SCF compared with subject with normal coronary flow (8.8 ± 0.8 vs. 5.4 ± 0.4pg/ml, p b 0.01).
We evaluated the correlations of TIMI frame count among each major epicardial coronary arteries in patients with SCF. The data indicated that TIMI frame counts for each major epicardial coronary arteries were detected to be significantly correlated with each other (LAD-CX: γ = 0.841, p b 0.001; LAD-RCA: γ = 0.892, p b 0.001; CX-RCA: γ = 0.785, p b 0.001).
Table 2 TIMI frame count and vessel diameter (mean ± SD) Variables
SCF group
NCF group
(n = 42)
(n = 30)
p value
Vessel diameter LAD (mm) LCX (mm) RCA (mm) Mean (mm)
3.02 ± 0.36 2.84 ± 0.32 3.06 ± 0.5 3.03 ± 0.20
3.00 ± 0.31 2.87 ± 0.40 3.02 ± 0.32 3.01 ± 0.21
NS NS NS NS
TIMI frame count LAD LCX RCA Mean
65.8 ± 18.2 42.1 ± 13.7 46.8 ± 15.3 47.6 ± 16.2
39.2 ± 4.5 20.0 ± 3.5 21.6 ± 2.5 28.2 ± 3.0
b0.01 b0.01 b0.01 b0.01
TIMI = Thrombolysis In Myocardial Infarction; LAD = left anterior descending artery; LCX = left circumflex artery; RCA = right coronary artery; NS = not significance.
3.5. Correlation between inflammatory markers and TIMI frame counts In addition, the analysis of correlation between inflammatory markers and TIMI frame count was also performed in patients with SCF. We found a significantly positive correlation between inflammatory markers including CRP and IL-6 and TIMI frame counts (CRP: γ = 0.551, p b 0.01; IL-6: γ = 0.573, p b 0.01). 4. Discussion In the present study, we demonstrated that increased inflammatory markers including CRP and IL-6 were found in patients with SCF. Moreover, in patients with SCF, a significant positive correlation was found between increased inflammatory markers and TIMI frame count in the study. This might be a suggestion that low-grade, chronic inflammation may be involved in SCF. In fact, SCF phenomenon was first described in 1972 [1]. However, since that time, only a limited number of studies have
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J.-J. Li et al. / Clinica Chimica Acta 385 (2007) 43–47
focused on the etiology of this unique angiographic phenomenon. Although the pathophysiological mechanisms of SCF remain uncertain, there are several hypotheses have been suggested. Occlusive disease of small coronary arteries has been suggested as an etiology of SCF [13]. Based on this hypothesis, SCF phenomenon may be a form of early phase of atherosclerosis in some patients [14]. In addition, the small vessel dysfunction has been typically implicated in the pathogenesis of SCF since its first description [15]. The evidence of affliction of small vessels comes from the results of histopathological examination of ventricular biopsy specimens in patients with SCF. Mosseri et al. reported abnormalities of small coronary arteries along with myocardial hypertrophy and patchy fibrosis in the biopsy samples from right ventricle of six patients with SCF [4]. However, majority of these patients had concomitant diseases that could have induced these changes. Later, Mangieri et al. reported histopathological examination of left ventricular endomyocardial biopsy specimens in a more homogenous group of 10 patients of SCF who did not have any other cardiac or systemic diseases [3]. Moreover, the mechanism of the imbalance between vasoconstrictor and vasodilatory factors has been also proposed for SCF. Recent published studies have highlighted the imbalance between endothelin-1 and nitric oxide release in patients with SCF as compared to controls with normal coronary flow [16–18]. Furthermore, the platelet function disorder has also been suggested to be involved in the pathogenesis of SCF [19,20]. The main finding emerging from Gokce et al. showed that the ratio of platelet aggregability was significantly higher in the patients with SCF than that in the control subjects, suggesting that platelet function disorder may be one of explanation for the pathogenesis of the underlying mechanism in SCF [20]. Inflammation has been reported to be a major contributing factor to many cardiovascular event, and demonstrated to be associated with different clinical settings of coronary artery disease. More recently, inflammation mechanism has also been suggested to be involved in SCF phenomenon. Turhan et al. performed a study to evaluating plasma soluble adhesion molecules; intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin as possible indicators of endothelial activation or inflammation in patients with SCF, but with angiographically proven normal coronary arteries in all three coronary vessels [21]. Coronary flow rate of all patients and control subjects were documented by TIMI frame count. The results showed that serum ICAM-1, VCAM-1, and E-selectin concentrations of patients with SCF were found to be significantly higher than those of control subjects with normal coronary flow. Average TIMI frame counts were detected to be significantly correlated with plasma soluble ICAM-1, VCAM-1 and E-selectin concentrations. Consistent with this previous study, we found that increased plasma inflammatory markers including CRP and IL-6 existed in patients with SCF in the present study. In addition, our data also showed that TIMI frame counts were significantly correlated with inflammatory markers, suggesting that an inflammation may be a contributor for the development of SCF (Table 3). In other word, increased concentrations of inflammatory markers in patients with SCF may be an indicator of endothelial
Table 3 Plasma concentrations of inflammatory markers in patients with slow coronary flow (mean ± SD) Variables
SCF group
NCF group
(n = 42)
(n = 30)
Hs-CRP (mg/l) Log-(hs-CRP) IL-6 (pg/ml)
2.8 [1.4–5.8] 0.27 ± 0.16 8.8 ± 0.8
1.7 [0.9–4.2] 0.22 ± 0.11 5.4 ± 0.4
p value b0.05 b0.01 b0.01
Hs-CRP = high-sensitivity C-reactive protein; IL-6 = interleukin-6.
activation and inflammation and are likely to be in the causal pathway involving in SCF. Despite the relative good prognosis of patients with SCF, the chronic, frequent nature of the persistent chest uncomfortable can significantly impair quality of life [22,23]. Therefore, an attention should be paid to this unique phenomenon. However, whether increased inflammatory markers are related to the pathogenesis of SCF in these patients deserved further investigation. 4.1. Limitations The small-sampled size may be a limitation in the present study. With the causes of an increased inflammatory marker in patients with SCF are unknown. However, it is warranted to investigate whether inflammation is involved in the pathogenesis of the underlying mechanism in SCF. Acknowledgements This article is partly supported by a Fu Wai Hospital Grant (2004190), National Natural Scientific Foundation (30670861), and Specialized Research Fund for the Doctoral Program of Higher Education of China (20060023044) awarded to Dr. JianJun Li, MD, PhD. References [1] Tambe AA, Demany MA, Zimmerman, et al. Angina pectoris and slow flow velocity of dye in coronary arteries-a new angiographic finding. Am Heart J 1972;84:66–71. [2] Goel K, Gupta SK, Agarwal, et al. Slow coronary flow: a distinct angiographic subgroup in syndrome X. Angiology 2001;52:507–14. [3] Mangieri E, Macchiarelli Ciavolella M, et al. Slow coronary flow: clinical and histopathological features in patients with otherwise normal epicardial coronary arteries. Catheter Cardiovasc Diagn 1996;37:375–81. [4] Mosseri M, Yarom R, Gotsman MS, et al. Histologic evidence for small vessel coronary artery disease in patients with angina pectoris and patent large coronary arteries. Circulation 1986;74:964–72. [5] Li J-J. Atheroscleritis is a more rational term for the pathological entity currently known as atherosclerosis. Med Hypotheses 2004;63:100–2. [6] Li J-J, Fang C-H. C-reactive protein is not only an inflammatory marker but also a direct cause of cardiovascular disease. Med Hypotheses 2004;62:499–506. [7] Shah PK. Inflammation, neointimal hyperplasia, and restenosis: as the leukocytes roll, the arteries thicken. Circulation 2003;107:2175–7. [8] Li J-J. Inflammation: an important mechanism for different clinical entities of coronary artery disease. Chin Med J 2005;118:1817–26. [9] Li J-J, Xu B, Li Z-C, et al. Is slow coronary flow associated with inflammation. Med Hypotheses 2006;66:527–30. [10] Li J-J, Wang H-R, Huang C-X, et al. Enhanced response of blood monocytes to C-reactive protein in patients with unstable angina. Clin Chim Acta 2004;22(352):127–33.
J.-J. Li et al. / Clinica Chimica Acta 385 (2007) 43–47 [11] Li J-J, Chen X-J. Simvastatin inhibits interleukin-6 release in human monocytes stimulated by C-reactive protein and lipopolysaccharide. Coron Artery Dis 2003;14:329–34. [12] Gibson CM, Cannon CP, Daley WL, et al. TIMI frame count: a quantitative method of assessing coronary artery flow. Circulation 1996;93:879–88. [13] Mintz GS, Painter JA, Pichard AD, et al. Atherosclerosis in angiographically “normal” coronary artery reference segments: an intravascular ultrasound study with clinical correlations. J Am Coll Cardiol 1995;25:1479–85. [14] Pedkdemir H, Cin VG, Cicek D, et al. Slow coronary flow may be a sign of diffuse atherosclerosis: contribution of FFR and IVUS. Acta Cardiol 2004;59:127–33. [15] Kelly RF, Sompalli V, Sattar P, et al. Increased TIMI frame counts in cocaine users: a case for increased microvascular resistance in the absence of epicardial coronary disease or spasm. Clin Cardiol 2003;26:319–22. [16] Pekdemir H, Polat G, Cin G, et al. Increased plasma endothelin-1 concentrations in coronary sinus during rapid right atrial pacing in patients with slow coronary flow. Int J Cardiol 2004;97:35–41. [17] Camsari A, Pekdemir H, Cicek D, et al. Endothelin-1 and nitric oxide concentrations and their response to exercise in patients with slow coronary flow. Circ J 2003;67:1022–8.
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[18] Sezgin N, Barutcu I, Sezgin AT, et al. Plasma nitric oxide concentration and its role in slow coronary phenomenon. Int Heart J 2005;46:373–82. [19] Lanza GA, Andreotti F, Sestio A, et al. Platelet aggregability in cardiac syndrome X. Eur Heart J 2001;22:1924–30. [20] Gokce M, Kaplan S, Tekelioglu Y, et al. Platelet function disorder in patients with coronary slow flow. Clin Cardiol 2005;28:145–8. [21] Turhan H, Saydam GS, Erbay AR, et al. Increased plasma soluble adhesion molecules: ICAM-1, VCAM-1, and E-selectin concentrations in patients with slow coronary flow. Int J Cardiol 2005;98:450–6. [22] Burchartt BA, Mukeriji V, Alpert MA, et al. Coronary artery slow flow associated with angina pectoris and hypertension: a case report. Angiology 1998;49:483–7. [23] Przybojewski JZ, Becker PH. Angina pectoris and acute myocardial infarction due to “slow-flow phenomenon” in nonatherosclerotic coronary arteries: a case report. Angiology 1986;37:751–61.
Increased plasma C-reactive protein and interleukin-6 concentrations in patients with slow coronary flow Jian-Jun Li ⁎, Xue-Wen Qin, Zi-Cheng Li 1 , He-Song Zeng 2 , Zhan Gao, Bo Xu, Chao-Yang Zhang, Jie Li Department of Cardiology, Fu Wai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100037, People's Republic of China Received 16 June 2006; received in revised form 30 May 2007; accepted 31 May 2007 Available online 15 June 2007
Abstract Background: Slow coronary filling of epicardial coronary arteries in the absence of stenosis is not infrequently detected finding during routine coronary angiography. There is mounting evidence suggested that an inflammatory process play an important role in atherosclerotic pathogenesis appeared in different clinical settings. However, the possible association between inflammation and slow coronary flow (SCF) has not been investigated. We examined whether the increased inflammatory markers are present in patients with SCF. Methods: Forty-two patients with SCF detected by coronary angiography via the Thrombosis In Myocardial Infarction (TIMI) frame count method were enrolled in this study. The plasma concentration of high-sensitivity C-reactive protein (CRP) and interleukin-6 (IL-6) were evaluated using commercial available kits. Data were compared with 30 control subjects with angiographically normal coronary flow. Results: There are no differences regarding clinical characteristics between the 2 groups. The data showed, however, that plasma CRP and IL-6 concentrations were higher in patients with SCF compared with normal control subject (CRP: 0.27 ± 0.16 vs. 0.22 ± 0.11mg/l; and IL-6: 8.7 ± 0.8 vs. 5.4 ± 0.4pg/ml, p b 0.01 respectively). In addition, mean TIMI frame count was positively correlated with plasma CRP and IL-6 concentrations (CRP: γ = 0.551; IL-6: γ = 0.573, p b 0.01 respectively). Conclusions: Plasma concentration of CRP and IL-6 concentrations increased, and was positive correlated with TIMI frame count in patients with SCF compared with normal coronary flow subject. Therefore, whether the increased inflammatory markers are related to the pathogenesis of SCF in these patients deserved further investigation. © 2007 Elsevier B.V. All rights reserved. Keywords: Inflammation, C-reactive protein, Interleukin-6, Slow coronary flow
1. Introduction The slow coronary flow (SCF) phenomenon is an angiographic observation characterized by angiographically normal or near-normal coronary arteries with delayed opacification of the distal vasculature [1–3]. It has been reported that coronary endothelial dysfunction play an important pathogenetic role in patients with SCF. Moreover, myocardial biopsy studies have also revealed the presence of coronary microvascular disease in ⁎ Corresponding author. Tel.: +86 10 88396077; fax: +86 10 68331730. E-mail address: [email protected] (J.-J. Li). 1 Division of Cardiology, First Affiliated Hospital, Jinan University, Guangzhou 510630, PR China. 2 Cardiovascular Division, Tongji Hospital of Tongji Medical College, Huazhong University, of Science and Technology, Wuhan 430030, PR China. 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.05.024
patients exhibiting SCF [4]. However, the precise mechanisms responsible for this microvascular endothelial dysfunction in patients with SCF are still unknown. Although atherosclerosis has been considered to be multifactorial disease in which genetic, environmental, metabolic factors have been implicated, the gaps remain in our knowledge of the etiopathogenesis of atherosclerosis. There is mounting evidence that inflammation plays an important role in the initiation, development as well as evolution of atherosclerosis, suggesting that atherosclerosis is an inflammation disease [5–8]. Although triggers and pathways of inflammation are probably multiple and different in different clinical settings, the data from animals as well as humans including our groups indicated that an inflammatory process was involved in all stages of atherosclerosis appeared in different clinical entities [9–11].
44
J.-J. Li et al. / Clinica Chimica Acta 385 (2007) 43–47
In addition, a large amount of data indicated that C-reactive protein (CRP) is a sensitive marker of underlying systemic inflammation, are increased among men and women at risk for future cardiovascular events, and the addition of CRP testing to standard lipid screening seems to provide an improved method to determine vascular risk [6]. These data, as well as accumulating evidence that CRP may have direct inflammatory effects at the endothelial levels. Besides, among pro-inflammatory cytokines, interleukin-6 (IL-6) is one of the most important factors and has multiple important effects in human pathphysiology. In our previous study, the data showed that an enhanced production of IL-6 by monocytes in response to CRP in patients with unstable angina [10]. Moreover, IL-6 is a powerful stimulus for CRP production [11]. Based on those observations, we hypothesis that an inflammatory process may be associated with the development of SCF [9]. 2. Methods 2.1. Subjects The protocol of the study were approved by the Ethics Review Board of Fu Wai Hospital, Chinese Academy of Medical Science and Peking Union Medical College, and Tongji Hospital of Tongji Medical College, Huazahong University of Science and Technology, and First Affiliated Hospital of Jinan University, and all the patients provided informed consent. The study groups included 42 consecutive patients referred for assessment to our centers for Diagnosis and Treatment of Coronary Artery Disease. Entry criteria were patients with angiographically proven normal coronary arteries and show SCF in all 3 coronary vessels (SCF group, 34 males and 8 females, mean age 49 ± 7y), and 30 subjects with angiographically proven normal coronary artery with normal coronary flow (NCF group, 25 males and 5 females, mean age 50 ± 8y). All patients with SCF were selected from individuals who underwent coronary angiography in our centers with a suspicion of coronary artery disease and diagnosed as having angiographically normal coronary arteries. Control subjects consisted of 30 consecutive subjects with atypical chest pain admitted to our center or division for selective coronary angiography and subsequently found to have angiographically normal coronary arteries with normal coronary flow. All subjects enrolled in this study had normal hepatic and renal function. The hyperlipidemia was defined as low-density lipoprotein cholesterol ≥ 160mg/dl or total cholesterol ≥ 220mg/dl and/or triglyceride (TG) ≥ 200mg/dl. Patients with evidence of coronary artery disease, myocardial infarction, valvular heart disease, congestive heart failure, left ventricular dysfunction, echocardiographically proven left ventricular hypertrophy, a history of dysphagia, swallowing as well as intestinal motility disorders, untreated thyroid disease, sinus node dysfunction or conduction disturbance, estrogen replacement therapy, carcinoma, poorly controlled hypertension (systolic blood pressure N 160mm Hg or diastolic blood pressure N 105mm Hg), recent major operation (b 3months), autoimmune disease, metabolic syndrome and infection were excluded from the study.
2.2. Coronary angiography Left ventricular and selective coronary angiography for all enrolled patients was performed using the standard Judkin's techniques, and the results were analyzed by at least 2 interventional physicians according to our previous study. Only angiograms with visually smooth contours with no wall irregularities were considered as normal. Contrast used in angiography of this study is Iopromide (Ultravist-370, Schering AG, Berlin, Germany). Coronary flow rates of all subjects were determined by Thrombosis In Myocardial Infarction frame count (TIMI frame count) because the method is a simple, reproducible, objective and quantitative index of coronary flow velocity [12,13]. TIMI frame count was determined for each major coronary artery in each patient and control subject according to the method first described by Gibson et al. [12]. In brief, the number of cineangiographic frames, recorded at
30fps, required for the leading edge of the column of radiographic contrast to reach a predetermined landmark, is determined. The first frame is defined as the frame in which concentrated dye occupies the full width of the proximal coronary artery lumen, touching both borders of the lumen, and forward motion down the artery. The final frame is designated when the leading edge of the contrast column initially arrives at the distal landmark. In the left anterior descending (LAD) coronary artery, the landmark used is the most distal branch nearest the apex of the left ventricle, commonly referred as the “pitchfork”. All anti-anginal and anti-ischemic medications, except sublingual nitroglycerin, were withheld for at least 24h before the examination. During coronary angiography, to exclude the possibility of coronary spasm, all patients underwent hyperventilation tests, which were performed by asking the patients to breathe quickly and deeply for at least 5min.
2.3. CRP and IL-6 determinations EDTA-anticoagulated peripheral blood sample were taken after 12-h overnight fast at baseline (before coronary angiography). The plasma was obtained after a centrifugation of 3000rpm at 4°C for 15min. The concentrations of high-sensitivity CRP were determined using immunoturbidometry (Beckmann Assay 360) as our previously reported [10]. The median normal value for CRP is 0.8mg/l, with 90% of normal values b 0.3mg/l, with a lower detection limit of 0.2mg/l. The inter-assay CVs were 4.4% and 4.8%, respectively, and intra-assay CVs were 3.5% and 5.1%, respectively. IL-6 was measured with a commercial assay kit (Quantikine human IL-6, R & D System). IL-6 measurements were performed from plasma in duplicate, and both intra- and inter-assay CV was b 10%. The range of values detected by the assay for IL-6 was 3 to 5000pg/ml as our previously reported [11].
2.4. Statistical analysis Continuous variables are expressed as mean ± SD, and categorical variables were expressed as percentage. Comparison of categorical and continuous variables between the two groups was performed using chi-square test and unpaired t test, respectively. Because the distribution of CRP is skewed rightward, log transformation was made at baseline, and the significance of any difference in distributions was assessed by the Wilcoxon rank–sum test as our previously reported [11]. The correlation among each major epicardial coronary arteries and between the plasma concentrations of log-CRP or IL-6 and mean TIMI frame count was assessed by the Pearson correlation test. A p b 0.05 was considered statistically significant.
Table 1 Baseline clinical characteristics (mean ± SD) Variables
SFC group
NCF group
p value
(n = 42)
(n = 30)
Age (y) Male/female Body mass index (kg/m2) Family history of CAD, n (%) Current smoker, n (%) Hypertension, n (%) Hperlipidemia, n (%) Diabetes, n (%) EF (%)
49 ± 7 34/8 23 ± 4 5 (12) 10 (24) 11 (26) 5 (12) 2 (5) 60 ± 8
50 ± 8 25/5 22 ± 4 2 (7) 6 (20) 5 (17) 4 (13) 1 (3) 59 ± 7
NS NS NS NS NS NS NS NS NS
Medications Aspirin, n (%) β-blocker, n (%) ACEI, n (%) Statin, n (%) CCB, n (%)
42 (100) 27 (64) 10 (24) 5 (12) 4 (10)
30 (100) 21 (70) 3 (10) 4 (13) 2 (7)
NS NS NS NS NS
CAD = coronary artery disease; NS = not significance; ACEI = angiotensinconverting enzyme inhibitor; CCB = calcium channel blocker; EF = ejection fraction.
J.-J. Li et al. / Clinica Chimica Acta 385 (2007) 43–47
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3. Results 3.1. No difference of baseline clinical characteristics between the two groups Baseline clinical characteristics of subjects with slow coronary flow (n = 42) and with angiographically normal coronary flow (n = 30) are given in Table 1. There are no differences regarding clinical characteristics between the 2 groups including age, sex, body mass index, history of coronary artery disease, smoker, hypertension, hyperlipidemia, diabetes, and medications used before. 3.2. TIMI frame count and vessels diameter TIMI frame counts including LAD, left circumflex artery (LCX), right coronary artery (RCA) and vessels diameters are presented in Table 2. The data showed that patients with SCF were detected to have significantly higher TIMI frame count compared to normal control subjects for each major epicardial coronary artery. Mean TIMI frame count was found to be significantly higher in patients with SCF compared with normal control subjects (47.6 ± 16.2 vs 28.2 ± 3.0, p b 0.01). However, mean epicardial coronary artery diameters were detected to be similar in patients with SCF and normal control subjects (3.03 ± 0.20 vs. 3.01 ± 0.21mm, p N 0.05).
Fig. 1. Comparison of mean CRP (A) and IL-6 (B) between slow coronary flow group (SCF group, n = 42) and normal coronary flow group (NCF group, n = 30). Data are mean ± S.D. ⁎p b 0.01 compared with NCF group. CRP = C-reactive protein; IL-6 = interleukin-6.
3.3. Increased CRP and IL-6 concentrations in patients with SCF
3.4. Correlation among each major epicardial coronary arteries
As showed in Table 2 and Fig. 1A and B, the plasma CRP concentrations were higher in patients with SCF than that in subjects with normal coronary flow (0.27 ± 0.16 vs. 0.22 ± 0.11mg/l, p b 0.01). In addition, the pattern of plasma IL-6 concentrations was also similar as CRP concentrations. That is, the increased plasma IL-6 concentration was also found in patients with SCF compared with subject with normal coronary flow (8.8 ± 0.8 vs. 5.4 ± 0.4pg/ml, p b 0.01).
We evaluated the correlations of TIMI frame count among each major epicardial coronary arteries in patients with SCF. The data indicated that TIMI frame counts for each major epicardial coronary arteries were detected to be significantly correlated with each other (LAD-CX: γ = 0.841, p b 0.001; LAD-RCA: γ = 0.892, p b 0.001; CX-RCA: γ = 0.785, p b 0.001).
Table 2 TIMI frame count and vessel diameter (mean ± SD) Variables
SCF group
NCF group
(n = 42)
(n = 30)
p value
Vessel diameter LAD (mm) LCX (mm) RCA (mm) Mean (mm)
3.02 ± 0.36 2.84 ± 0.32 3.06 ± 0.5 3.03 ± 0.20
3.00 ± 0.31 2.87 ± 0.40 3.02 ± 0.32 3.01 ± 0.21
NS NS NS NS
TIMI frame count LAD LCX RCA Mean
65.8 ± 18.2 42.1 ± 13.7 46.8 ± 15.3 47.6 ± 16.2
39.2 ± 4.5 20.0 ± 3.5 21.6 ± 2.5 28.2 ± 3.0
b0.01 b0.01 b0.01 b0.01
TIMI = Thrombolysis In Myocardial Infarction; LAD = left anterior descending artery; LCX = left circumflex artery; RCA = right coronary artery; NS = not significance.
3.5. Correlation between inflammatory markers and TIMI frame counts In addition, the analysis of correlation between inflammatory markers and TIMI frame count was also performed in patients with SCF. We found a significantly positive correlation between inflammatory markers including CRP and IL-6 and TIMI frame counts (CRP: γ = 0.551, p b 0.01; IL-6: γ = 0.573, p b 0.01). 4. Discussion In the present study, we demonstrated that increased inflammatory markers including CRP and IL-6 were found in patients with SCF. Moreover, in patients with SCF, a significant positive correlation was found between increased inflammatory markers and TIMI frame count in the study. This might be a suggestion that low-grade, chronic inflammation may be involved in SCF. In fact, SCF phenomenon was first described in 1972 [1]. However, since that time, only a limited number of studies have
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J.-J. Li et al. / Clinica Chimica Acta 385 (2007) 43–47
focused on the etiology of this unique angiographic phenomenon. Although the pathophysiological mechanisms of SCF remain uncertain, there are several hypotheses have been suggested. Occlusive disease of small coronary arteries has been suggested as an etiology of SCF [13]. Based on this hypothesis, SCF phenomenon may be a form of early phase of atherosclerosis in some patients [14]. In addition, the small vessel dysfunction has been typically implicated in the pathogenesis of SCF since its first description [15]. The evidence of affliction of small vessels comes from the results of histopathological examination of ventricular biopsy specimens in patients with SCF. Mosseri et al. reported abnormalities of small coronary arteries along with myocardial hypertrophy and patchy fibrosis in the biopsy samples from right ventricle of six patients with SCF [4]. However, majority of these patients had concomitant diseases that could have induced these changes. Later, Mangieri et al. reported histopathological examination of left ventricular endomyocardial biopsy specimens in a more homogenous group of 10 patients of SCF who did not have any other cardiac or systemic diseases [3]. Moreover, the mechanism of the imbalance between vasoconstrictor and vasodilatory factors has been also proposed for SCF. Recent published studies have highlighted the imbalance between endothelin-1 and nitric oxide release in patients with SCF as compared to controls with normal coronary flow [16–18]. Furthermore, the platelet function disorder has also been suggested to be involved in the pathogenesis of SCF [19,20]. The main finding emerging from Gokce et al. showed that the ratio of platelet aggregability was significantly higher in the patients with SCF than that in the control subjects, suggesting that platelet function disorder may be one of explanation for the pathogenesis of the underlying mechanism in SCF [20]. Inflammation has been reported to be a major contributing factor to many cardiovascular event, and demonstrated to be associated with different clinical settings of coronary artery disease. More recently, inflammation mechanism has also been suggested to be involved in SCF phenomenon. Turhan et al. performed a study to evaluating plasma soluble adhesion molecules; intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin as possible indicators of endothelial activation or inflammation in patients with SCF, but with angiographically proven normal coronary arteries in all three coronary vessels [21]. Coronary flow rate of all patients and control subjects were documented by TIMI frame count. The results showed that serum ICAM-1, VCAM-1, and E-selectin concentrations of patients with SCF were found to be significantly higher than those of control subjects with normal coronary flow. Average TIMI frame counts were detected to be significantly correlated with plasma soluble ICAM-1, VCAM-1 and E-selectin concentrations. Consistent with this previous study, we found that increased plasma inflammatory markers including CRP and IL-6 existed in patients with SCF in the present study. In addition, our data also showed that TIMI frame counts were significantly correlated with inflammatory markers, suggesting that an inflammation may be a contributor for the development of SCF (Table 3). In other word, increased concentrations of inflammatory markers in patients with SCF may be an indicator of endothelial
Table 3 Plasma concentrations of inflammatory markers in patients with slow coronary flow (mean ± SD) Variables
SCF group
NCF group
(n = 42)
(n = 30)
Hs-CRP (mg/l) Log-(hs-CRP) IL-6 (pg/ml)
2.8 [1.4–5.8] 0.27 ± 0.16 8.8 ± 0.8
1.7 [0.9–4.2] 0.22 ± 0.11 5.4 ± 0.4
p value b0.05 b0.01 b0.01
Hs-CRP = high-sensitivity C-reactive protein; IL-6 = interleukin-6.
activation and inflammation and are likely to be in the causal pathway involving in SCF. Despite the relative good prognosis of patients with SCF, the chronic, frequent nature of the persistent chest uncomfortable can significantly impair quality of life [22,23]. Therefore, an attention should be paid to this unique phenomenon. However, whether increased inflammatory markers are related to the pathogenesis of SCF in these patients deserved further investigation. 4.1. Limitations The small-sampled size may be a limitation in the present study. With the causes of an increased inflammatory marker in patients with SCF are unknown. However, it is warranted to investigate whether inflammation is involved in the pathogenesis of the underlying mechanism in SCF. Acknowledgements This article is partly supported by a Fu Wai Hospital Grant (2004190), National Natural Scientific Foundation (30670861), and Specialized Research Fund for the Doctoral Program of Higher Education of China (20060023044) awarded to Dr. JianJun Li, MD, PhD. References [1] Tambe AA, Demany MA, Zimmerman, et al. Angina pectoris and slow flow velocity of dye in coronary arteries-a new angiographic finding. Am Heart J 1972;84:66–71. [2] Goel K, Gupta SK, Agarwal, et al. Slow coronary flow: a distinct angiographic subgroup in syndrome X. Angiology 2001;52:507–14. [3] Mangieri E, Macchiarelli Ciavolella M, et al. Slow coronary flow: clinical and histopathological features in patients with otherwise normal epicardial coronary arteries. Catheter Cardiovasc Diagn 1996;37:375–81. [4] Mosseri M, Yarom R, Gotsman MS, et al. Histologic evidence for small vessel coronary artery disease in patients with angina pectoris and patent large coronary arteries. Circulation 1986;74:964–72. [5] Li J-J. Atheroscleritis is a more rational term for the pathological entity currently known as atherosclerosis. Med Hypotheses 2004;63:100–2. [6] Li J-J, Fang C-H. C-reactive protein is not only an inflammatory marker but also a direct cause of cardiovascular disease. Med Hypotheses 2004;62:499–506. [7] Shah PK. Inflammation, neointimal hyperplasia, and restenosis: as the leukocytes roll, the arteries thicken. Circulation 2003;107:2175–7. [8] Li J-J. Inflammation: an important mechanism for different clinical entities of coronary artery disease. Chin Med J 2005;118:1817–26. [9] Li J-J, Xu B, Li Z-C, et al. Is slow coronary flow associated with inflammation. Med Hypotheses 2006;66:527–30. [10] Li J-J, Wang H-R, Huang C-X, et al. Enhanced response of blood monocytes to C-reactive protein in patients with unstable angina. Clin Chim Acta 2004;22(352):127–33.
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