Key words: coronary artery calcification; C-reactive protein; electron beam CT; endothelial function

Enhanced Coronary Calcification Determined by Electron Beam CT Is Strongly Related to Endothelial Dysfunction in Patients With Suspected Coronary Artery Disease* Po-Hsun Huang, MD; Lung-Ching Chen, MD; Hsin-Bang Leu, MD; Philip Yu-An Ding, MD, PhD; Jaw-Wen Chen, MD; Tao-Cheng Wu, MD; and Shing-Jong Lin, MD, PhD Background: Coronary artery calcification determined by electron beam CT (EBCT) is strongly associated with total plaque burden but is not related to systemic vascular inflammation. Aims: We sought to test the hypothesis that enhanced coronary artery calcification, a marker of atherosclerosis and plaque burden, was related to endothelial dysfunction in patients with suspected coronary artery disease (CAD). Methods and results: One hundred twenty-four subjects with suspected CAD were enrolled. Coronary artery calcification was detected by EBCT. A noninvasive method of brachial ultrasound was used to measure endothelium-dependent flow-mediated vasodilation (FMD) and endothelium-independent nitroglycerin-mediated vasodilation (NMD). Serum high-sensitivity C-reactive protein (hscrp) and monocyte chemoattractant protein-1 (MCP-1) levels were also determined. Of the 124 patients, the calcium scores ranged from 0 to 4,394. All subjects were classified into three groups according to coronary calcium scores: group 1, score 0 (n 26); group 2, scores 1 to 199 (n 50); group 3, scores > 200 (n 48). There was an inverse association between the degree of coronary artery calcification and the endothelium-dependent FMD in the three groups (6.9 0.6% vs 5.3 0.3% vs 3.7 0.3%, respectively; p < 0.001) but not the endothelium-independent NMD. Besides, no significant difference in serum levels of hscrp and MCP-1 were found among the three groups. However, both the serum levels of hscrp and MCP-1 were correlated significantly with endothelium-dependent FMD (r 0.211, p 0.019; and r 0.188, p 0.037, respectively). By multivariate analysis, enhanced coronary calcification was a strong independent predictor of endothelial dysfunction (p < 0.001). Conclusion: Enhanced coronary artery calcification strongly predicted endothelial dysfunction in patients with suspected CAD. Also, serum levels of hscrp and MCP-1 were significantly correlated with endothelial function. These findings suggested that both calcium deposition and inflammation were involved in endothelial dysfunction. (CHEST 2005; 128:810 815) Key words: coronary artery calcification; C-reactive protein; electron beam CT; endothelial function Abbreviations: CAD coronary artery disease; CCS coronary calcium score; EBCT electron beam CT; FMD flow-mediated vasodilation; hscrp high-sensitivity C-reactive protein; MCP-1 monocyte chemoattractant protein-1; NMD nitroglycerine-mediated vasodilation *From the Division of Cardiology (Drs. Huang, L-C Chen, Leu, and Ding), Department of Internal Medicine, Taipei Veterans General Hospital; Institute of Clinical Medicine (Dr. Wu); and Cardiovascular Research Center (Drs. J-W Chen and Lin), National Yang-Ming University, Taipei, Taiwan. This study was partly supported by research grants NSC 92 2314-B-010 009 from the National Science Councils, VGH-239 from Taipei Veterans General Hospital, and CI 92 7-1 from the Yen Tjing Ling Medical Foundation, Taipei, Taiwan. Coronary artery calcification has been shown to play an important role in the development of atherosclerosis and is strongly associated with the total plaque burden proved in previous histopathologic studies. 1 3 The use of electron beam CT (EBCT) for accurate quantitative measurements has led to an increased interest in understanding the clinical importance of coronary calcium. 4 The coronary calcium score (CCS) determined by EBCT Manuscript received July 28, 2004; revision accepted February 1, 2005. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Shing-Jong Lin, MD, PhD, Division of Cardiology, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Rd, Taipei, Taiwan; e-mail: sjlin@vghtpe.gov.tw 810 Clinical Investigations

could provide useful prognostic information regarding subsequent coronary events in high-risk subjects with no clinical coronary artery disease (CAD), as well as in patients with the presence of CAD. 5,6 EBCT has also been demonstrated to be a useful technique for assessing the progression or regression of CAD in response to treatment of cardiovascular risk factors. 7 Endothelial dysfunction is thought to be an early sign of vascular endothelium injury and is the first critical step in the pathogenesis of atherosclerosis. Endothelial function plays a key role in determining the clinical manifestations of established atherosclerotic lesions and was proved predictive of further cardiovascular events. 8,9 However, no previous study has shown the relationship between the extent of coronary artery calcification and endothelial function. Therefore, in this study we test the hypothesis that enhanced CCSs determined by EBCT would be associated with endothelial dysfunction assessed by brachial ultrasonography. In addition, although conflicting results were shown about the relationship between C-reactive protein (CRP) and coronary calcification in asymptomatic patients, 10 12 in this study the associations of CRP and monocyte chemoattractant protein-1 (MCP-1) with coronary calcification and endothelial function were also evaluated in patients with suspected CAD. Study Population Materials and Methods The study population was composed of 124 consecutive patients with symptoms of typical or atypical chest pain who were referred for EBCT to determine coronary calcification between July 2002 and August 2003. We included patients who agreed to undergo brachial ultrasonography for endothelial function evaluation and blood sampling. Patients were excluded if they had history of previous cardiac surgery, myocardial infarction, heart failure, congenital heart disease, valvular heart disease, malignant hypertension, or significant endocrine, hepatic, or renal diseases. Before the study, a detailed review of each patient chart and an interview were conducted to gather data on symptoms, medications, coronary risk factors, previous cardiac events, smoking status, exercise habits, family history of CAD, and other systemic diseases. Blood biochemistry testing analyzed for lipid profiles, fasting sugar, uric acid, and creatinine. EBCT Scanning and Image Analysis EBCT scanning was performed (Imatron C-150XP Ultrafast CT scanner; Imatron; South San Francisco, CA) in the singleslice mode with an image-acquisition time of 100 ms and a section thickness of 3 mm. ECG triggering was used so that the image acquisition occurred after 80% of the R-R interval. Transverse image slices of the heart were obtained contiguously beginning 1 cm below the carina and progressing caudally through the apex to cover the whole heart. All obtained sections of the EBCT scan were then reviewed by an experienced radiologist who was blinded to all clinical data. The CCS for each artery was calculated by the radiologist using the methods of Agatston et al, as previously described. 4,13 Endothelium-Dependent Flow-Mediated Vasodilation Endothelium-dependent flow-mediated vasodilation (FMD) was assessed using a 7.5-MHz linear array transducer (Sonos 5500; Hewlett-Packard; Andover, MA) to scan the brachial artery in longitudinal section, as published previously. 14,15 All subjects were asked to fast, refrain from smoking, and withhold all medications for 12 h before the endothelial function test. To minimize mental stress, care was taken to make the patients as comfortable as possible, and the procedure was performed in a quiet air-conditioned room (22 to 25 C). The left arm was stabilized with a cushion, and a sphygmomanometric cuff was placed on the forearm. A baseline image was acquired, and blood flow was estimated by time averaging the pulsed Doppler velocity signals obtained from a midartery sample volume. Then the cuff was inflated to at least 50 mm Hg above systolic pressure to occlude arteries for 5 min and released abruptly. Postocclusion diameters were obtained at 60, 80, 100, and 120 s after deflation. FMD was calculated as the maximal postocclusion diameter relative to the averaged preocclusion diameters. A midartery pulsed Doppler signal was obtained immediately on cuff release and no later than 15 s after cuff deflation to assess hyperemic velocity. Endothelium-Independent Nitroglycerine-Mediated Vasodilation At least 10 min of rest was given after the reactive hyperemia before another image was acquired to reflect the reestablished baseline conditions. Diameter measurements were taken at least three times at 3- to 4-min intervals after 0.6-mg sublingual nitroglycerine administration. The maximal FMD and nitroglycerine-mediated vasodilation (NMD) diameters were determined as the average of the three consecutive maximal-diameter measurements after reactive hyperemia and nitroglycerine use, respectively. The FMD and NMD were then calculated as the percentage change in diameter compared with baseline. Serum Levels of High-Sensitivity CRP and MCP-1 After 12-h overnight fasting, blood samples were collected for measurement of high-sensitivity CRP (hscrp) and MCP-1. The blood samples were centrifuged at 3,000 revolutions per minute for 10 min immediately after collection, and then the serum samples were kept frozen at 70 C until analysis. Determination of hscrp levels was performed with use of latex-enhanced immunophelometric assay (Dade Behring; Marburg, Germany). MCP-1 in serum was quantified by a sandwich enzyme immunoassay technique (human MCP-1) [Quantikine TM; R&D Systems; Wiesbaden-Nordenstadt, Germany] according to the protocol of the manufacturer. Each standard and each serum sample were analyzed two times. The mean value was used for all subsequent analysis. Statistical Analysis All data are expressed as mean SEM; p 0.05 was considered to indicate statistical significance. Differences in baseline characteristics of underlying diseases, smoking status, exercise habits, medications were compared with the 2 test or Fisher Exact Test. Comparison among the three groups with FMD, www.chestjournal.org CHEST / 128 / 2/ AUGUST, 2005 811

Table 1 Baseline Characteristics of 124 Studied Patients in Three Groups* Characteristics Group 1 (CCS 0) n 26 Group 2 (CCS 1 199) n 50 Group 3 (CCS 200) n 48 p Value Age, yr 58 3 66 1 73 1 0.05 Male gender 14 (54) 29 (58) 35 (73) 0.175 Body mass index 23.6 0.4 26.0 0.6 24.1 0.5 0.05 Smoking status Current smokers 6 (23) 3 (6) 7 (15) 0.058 Never-smokers 18 (69) 31 (62) 26 (54) Former smokers 2 (8) 16 (16) 15 (31) Exercise habits 4 (15) 18 (36) 11 (23) 0.118 Family history of CAD 5 (19) 10 (20) 8 (17) 0.787 Systemic hypertension 7 (27) 26 (52) 31 (65) 0.05 Diabetes mellitus 2 (8) 9 (18) 15 (31) 0.05 Hypercholesterolemia (total cholesterol 10 (39) 19 (38) 13 (27) 0.447 200 mg/dl) Triglycerides, mg/dl 150 17 142 8 128 13 0.448 High-density lipoproteins, mg/dl 52 4 49 3 45 2 0.211 Fasting glucose, mg/dl 96 5 104 4 110 4 0.081 Uric acid, mg/dl 6.6 0.3 6.6 0.2 6.6 0.3 0.982 Creatinine, mg/dl 1.0 0.1 1.0 0.1 1.2 0.1 0.145 Positive treadmill exercise test or thallium scan results 16 (62) 30 (60) 28 (58) 0.656 *Data are presented as mean SEM or No. (%). NMD, hscrp, and MCP-1 were performed with analysis of variance and Scheffe test (post hoc test). Correlations between FMD, NMD, hscrp, and MCP-1 were calculated by Pearson correlation test. Multivariable analysis was analyzed by multiple linear regression method, and the variables included in the multivariate model were age, sex, body mass index, hypertension, diabetes mellitus, total cholesterol levels, high-density lipoproteins, and smoking status. Results A total of 124 patients were enrolled (78 men; 63%; mean age, 67 1 years); of these, 98 patients (79%) had positive EBCT results, ie, a CCS 0. All patients were classified into three groups according to CCSs: group 1, no calcification (CCS 0) [n 26]; group 2, small-to-moderate coronary calcification (CCS 1 to 199) [n 50]; and group 3, moderate-tosevere calcification (CCS 200) [n 48]. The baseline characteristics of the three groups are shown in Table 1. In group 3, the patients were older than those in groups 1 and 2 (p 0.05). No significant differences in baseline characteristics were found among the three groups, except there were more patients with hypertension and diabetes in group 3 than in groups 1 and 2 (p 0.05) and higher body mass index in group 2. There were more patients receiving medications, including antiplatelet agents, -blockers, and nitrates, in group 3 than in groups 1 and2(p 0.05), as shown in Table 2. Among the three groups, there was an inverse association between the extent of coronary artery calcification and endothelium-dependent FMD (6.9 0.6% vs 5.3 0.3% vs 3.7 0.3%, respectively; p 0.001), as shown in Table 3 and Figure 1, Table 2 Medications of 124 Studied Patients in Three Groups* Variables Group 1 (CCS 0) n 26 Group 2 (CCS 1 199) n 50 Group 3 (CCS 200) n 48 p Value Antiplatelet agents 6 (23) 30 (60) 38 (79) 0.05 -Blockers 7 (27) 20 (40) 27 (56) 0.05 Calcium-channel blockers 8 (31) 19 (38 26 (54) 0.103 Nitrates 6 (23) 14 (28) 23 (48) 0.05 -Blockers 1 (4) 5 (10) 9 (19) 0.144 Diuretics 0 (0) 9 (18) 8 (17) 0.072 Angiotensin-converting enzyme inhibitors 3 (12) 7 (14) 10 (21) 0.507 Angiotensin II receptor blockers 2 (8) 10 (20) 8 (17) 0.381 Statins 8 (31) 13 (26) 14 (29) 0.893 *Data are presented as No. (%). 812 Clinical Investigations

Table 3 Comparison of Baseline Parameters* Variables Group 1 (CCS 0) n 26 Group 2 (CCS 1 199) n 50 Group 3 (CCS 200) n 48 p Values Baseline diameter, mm 3.4 0.1 3.4 0.1 3.6 0.1 0.07 FMD, % 6.9 0.6 5.3 0.3 3.7 0.3 0.001 NMD, % 16.7 1.4 15.5 0.8 13.0 1.0 0.05 hscrp, mg/dl 1.5 0.4 2.2 0.4 2.7 0.4 0.219 MCP-1, pg/ml 206 19 229 13 216 13 0.547 *Data are presented as mean SEM. top, A. But no significant association existed between the CCS and endothelium-independent NMD (Fig 1, bottom, B). Both hscrp and MCP-1 revealed no significant difference among the three groups (p not significant), as shown in Table 3 and Figure 2. On the contrary, serum levels of hscrp and MCP-1 were shown to be correlated significantly with endotheliumdependent FMD (r 0.211, p 0.019; and r 0.188, p 0.037, respectively) but not with endothelium-independent NMD (p not significant). By multivariate analysis, enhanced coronary calcification was a strong independent predictor of endothelial dysfunction (p 0.001). Discussion To our knowledge, this is the first study to demonstrate that coronary calcification determined by EBCT is strongly related to endothelial dysfunction assessed by peripheral brachial ultrasonography in patients with suspected CAD. In addition, serum levels of hscrp and MCP-1, although not related to coronary calcification, were also found to be associated with endothelial function. Atherosclerotic calcification could begin as early as the second decade of life, just after fatty streak formation. 16,17 Calcium precipitates in diseased cor- Figure 1. The percentage changes of endothelium-dependent FMD (top, A) and endothelium-independent NMD (bottom, B) in the three patient groups. Figure 2. The percentage changes of hscrp (top, A) and MCP-1 (bottom, B) in the three patient groups. www.chestjournal.org CHEST / 128 / 2/ AUGUST, 2005 813

onary arteries by a mechanism similar to that found in active bone formation and remodeling. 18 Schmermund and colleagues 19 showed that the vast majority of patients with acute coronary syndromes and documented CAD have identifiable coronary calcium detected by EBCT. Coronary artery calcium is intimately associated with coronary atherosclerotic plaque development, but the presence or absence of calcium does not allow for reliable distinction between unstable vs stable plaque. 20 It can be speculated that coronary arterial calcification may represent an attempt to prevent acute coronary syndrome due to plaque rupture by strengthening weakened atherosclerotic plaque prone to rupture. During the past 2 decades, it has become evident that the vascular endothelium is a highly active organ that plays a key role in the regulation of vascular tone and maintenance of vascular homeostasis. 21,22 Endothelial dysfunction is characterized by a reduction of the bioavailability of vasodilators, particularly nitric oxide, and could predict further cardiovascular events. 23 25 Impaired FMD in the brachial artery has been proved to be closely correlated with endothelial dysfunction in patients with CAD. 26 In this study, enhanced coronary artery calcification were found to be significantly related to endothelial dysfunction in patients with suspected CAD, suggesting that the process of calcium precipitation in diseased coronary arteries is closely associated with endothelial dysfunction, an early sign of atherosclerosis. 27 Further studies are needed to clarify the exact interaction between calcium deposition and shear stress-stimulated nitric oxide production by endothelial cells. Increasing evidence suggests that atherosclerosis is an inflammatory disease. CRP, a serologic marker of inflammation, has been shown to be associated with the risk of future coronary events. 28 31 However, studies 10 12 of the relationship between CRP and coronary artery calcification in asymptomatic population did not reveal consistent results. The reasons for the lack of association between CRP and coronary artery calcification, in contrast to a more consistent association of CRP with clinical events, are not clear. Coronary arteries with higher calcium content may not necessarily exhibit more active inflammatory activity, as there may be several mechanisms responsible for the development of atherosclerosis. Park and coworkers 32 found that CCSs and plasma CRP levels provided incremental information regarding the risk of clinical cardiovascular events in asymptomatic, nondiabetic subjects in the South Bay Heart Watch Study. Both CRP and coronary calcification may reflect different mechanisms that lead to clinical events and provide additional benefits for risk stratification of CAD. The MCP-1, which exhibits its most potent activity toward monocytes, rapidly causes rolling monocytes to adhere firmly onto endothelial cells and subsequent infiltration into the subendothelial space. 33 36 In this study, MCP-1 was shown to be related to endothelial dysfunction. But lack of association between MCP-1 and coronary calcification again implies a different mechanism of involvement in atherosclerosis. In conclusion, we demonstrated that enhanced coronary artery calcification is significantly related to endothelium dysfunction in patients with suspected CAD but not related to hscrp or MCP-1. Endothelial dysfunction may play an important role in the process of coronary artery calcification. 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