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Vascular effects of diet and statin in hypercholesterolemic patients
International Journal of Cardiology 95 (2004) 185 – 191 www.elsevier.com/locate/ijcard
Vascular effects of diet and statin in hypercholesterolemic patients
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Kwang Kon Koh a,*, Ji Won Son a, Jeong Yeal Ahn b, Dong Kyu Jin a, Hyung Sik Kim c, Yu Mi Choi d, Tae Hoon Ahn a, Dae Sung Kim e, Eak Kyun Shin a a
Vascular Medicine and Atherosclerosis Unit, Division of Cardiology, Gil Heart Center, Gachon Medical School, 1198 Kuwol-dong, Namdong-gu, Incheon 405-760, South Korea b Clinical Pathology, Gachon Medical School, Namdong-gu, Incheon, South Korea c Radiology, Gachon Medical School, Namdong-gu, Incheon, South Korea d Nutrition, Gachon Medical School, Namdong-gu, Incheon, South Korea e Preventive Medicine (Biostatistics), Gachon Medical School, Namdong-gu, Incheon, South Korea Received 26 November 2002; received in revised form 5 May 2003; accepted 6 May 2003
Abstract Objective: We investigated whether statin improves nitric oxide (NO) bioactivity and reduces serological markers of oxidant stress and inflammation and whether statin-induced reduction in markers of oxidant stress and inflammation is mediated by improvement in NO bioactivity or lipoprotein changes, compared with American Heart Association Step I Diet (Diet). Methods: We administered diet + placebo and diet + simvastatin 20 mg daily during 14 weeks with randomized order to 31 and 32 patients with coronary artery disease, respectively, with a randomized design. Results: Compared with diet alone, simvastatin significantly improved the percent flow-mediated dilator response to hyperemia and lowered plasma levels of tumor necrosis factor (TNF)-a, intercellular adhesion molecule type-1 (ICAM-1), serum levels of CRP, and fibrinogen ( P < 0.001, P < 0.001, P = 0.035, P < 0.001 and P = 0.014, respectively). Compared with diet alone, simvastatin lowered but statistically insignificant plasma levels of nitrate and malondialdehyde (MDA) ( P = 0.164 and P = 0.150, respectively). Further, we observed that patients with the highest pretreatment TNF-a, ICAM-1, and CRP levels showed the greatest extent of reductions on simvastatin. There were significant inverse correlation between low-density lipoprotein (LDL) cholesterol or the ratio of LDL to HDL cholesterol levels and flow-mediated dilation percent (r = 0.342, P = 0.009 and r = 0.356, P = 0.006, respectively). Of interest, there were significant inverse correlations between flow-mediated dilation percent and TNF-a levels (r = 0.329, P = 0.010). However, no significant correlations between lipoprotein levels and levels of inflammation markers were determined. Despite the significant changes of lipoproteins, diet alone did not decrease the markers of inflammation. Conclusions: Compared with diet alone, simvastatin significantly reduced markers of inflammation more. These effects were independent of lipoprotein changes. D 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: HMG-CoA reductase inhibitor; Diet; Endothelial function; Oxidant stress; Inflammation; Atherosclerosis
1. Introduction Vascular inflammation plays an important role in the pathogenesis of atherosclerosis [1]. Inflammation of the cap and shoulder of the plaque is a common feature in atherosclerotic femoral and coronary arteries [2]. Indeed, increased plasma levels of inflammatory markers were correlated with the extent of disease [3]. Statins reduce levels of some $ We presented our study in part as abstracts in the American Heart Association 75th Annual Scientific Session in Chicago in November 17 – 20, 2002. * Corresponding author. Tel.: +82-32-460-3683; fax: +82-32-460-3117/ 467-9302. E-mail address: [email protected] (K.K. Koh).
0167-5273/$ - see front matter D 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2003.05.018
markers of inflammation (e.g. C-reactive protein, CRP) when administered to hypercholesterolemic subjects, in a manner that appears to be independent of lipid-lowering effects [4]. Endothelial dysfunction of epicardial coronary arteries precedes development of atherosclerotic disease that is either angiographically apparent or of sufficient obstructive severity to cause myocardial ischemia and angina pectoris [5]. Patients with coronary heart disease or risk factors for coronary heart disease have been associated with impaired functions of the endothelium. The vessel wall in these conditions may promote inflammation, which contributes to development and clinical expression of atherosclerosis [1]. Statins improve endothelial function, consistent with enhanced nitric oxide (NO) bioactivity [6– 8].
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NO may protect nuclear transcription factor, NFnB, from activation by oxidized low-density lipoprotein (LDL) or cytokines, and thus prevent or attenuate the transcription and expression of adhesion molecules [9]. Thus, therapies that increase NO bioactivity may reduce synthesis of proinflammatory proteins on the endothelial cell surface, which may reduce inflammation. In this regard, we have previously shown that unopposed conjugated equine estrogen improved endothelium-dependent vasodilator responsiveness, an index of NO bioactivity and reduced adhesion molecules in healthy or hypercholesterolemic post-menopausal women [6,10]. Statins improved NO bioactivity in humans [6– 8] and reduced inflammatory cells within atherosclerotic plaque in experimental models [11]. However, recent studies suggest that the beneficial effects of statins on clinical events may involve non-lipid mechanisms that affect endothelial function: inflammatory responses, thrombus formation, and plaque stability [1,12,13]. The purpose of this study is to determine: (1) whether statin reduces serological markers of oxidant stress and inflammation potentially affected by NO-potentiating properties, (2) whether statin-induced reduction in markers of inflammation is mediated by improvement in NO bioactivity or lipoprotein changes, compared with Step I Diet, (3) the mechanism of CRP and other cytokines regulation because CRP induces the synthesis of cell adhesion molecules in monocytes and endothelial cells [14].
and h-blocker therapy on a long-term basis. The study was approved by the Gil Hospital Institute Review Board and all participants gave written, informed consent.
2. Methods
2.3. Vascular studies
2.1. Study population and design
Imaging studies of the right brachial artery were performed using a ATL HDI 3000 ultrasound machine equipped with a 10 MHz linear-array transducer, based on a previously published technique [6,10]. All images were transmitted to a personal computer via Ethernet with Digital Imaging and Communication in Medicine (DICOM) format and then saved on the hard disk of personal computer as a BMP format. Arterial diameters were measured with Image ToolR for Windows version 2.0 (University of Texas Health Science Center, San Antonio, TX). Measurements were performed by two independent investigators (D.K.J. and H.S.K.) blinded to the subject’s identity and medication status. Measurements of maximum diameter and percent flow-mediated dilation were made in 10 studies selected at random. The interobserver and intraobserver variability for repeated measurement of maximum diameter were 0.004 F 0.039 and 0.005 F 0.089 mm, respectively. The interobserver and intraobserver variability for repeated measurement of percent flow-mediated dilation were 0.07 F 1.27% and 0.15 F 1.24%, respectively.
Sixty-three patients with angiographically-documented coronary artery disease were enrolled in this study. We excluded patients with acute myocardial infarction or unstable angina in order to avoid the effects of acute ischemic injury on inflammatory process. All patients were in Canadian Cardiovascular Society class I or II. All patients were taught and placed on American Heart Association Step I Diet [14-1] through the study period. We administered diet + placebo and diet + simvastatin 20 mg daily during 14 weeks with randomized order to 31 and 32 patients with coronary artery disease, respectively, with a randomized design. Both groups were age- and sex-matched. The mean age of patients were 62-year-old and the number of male were 13 among 32. The risk factors were hypertension (68% vs. 72%; diet + placebo vs. diet + simvastatin), diabetes (23% vs. 25%), and current smoking (35% vs. 31%). The proportion of medications were h adrenergic blockers (81% vs. 81%), calcium channel blockers (48% vs. 47%), angiotensin converting enzyme inhibitors (23% vs. 28%), long-acting nitrates (81% vs. 84%), and aspirin (84% vs. 88%). Vasoactive medications, including calcium channel blockers, angiotensin converting enzyme inhibitors, and long-acting nitrates, were withheld for z 24 h before the study. All patients were taking aspirin
2.2. Laboratory assays Blood samples for laboratory assays were obtained at approximately 08:00 h. following overnight fasting before and after treatment for 14 weeks and immediately coded so that investigators performing laboratory assays were blinded to subject identity or study sequence. Assays for lipids, plasma nitrate (using the Griess reaction), malondialdehyde (MDA), E-selectin, intercellular adhesion molecule type-1 (ICAM-1), vascular cell adhesion molecule type-1 (VCAM1), and tumor necrosis factor (TNF)-a were performed in duplicate by ELISA (R&D Systems and BIOXYTECHR LPO-586, OxisResearch) as previously described [6,10,15]. Fibrinogen was measured by the thrombin-time titration method (Thrombocheck FibR, Japan). In all patients, serum was collected for the measurement of CRP levels. CRP levels were determined with an immunonephelometry system according to methods described by the manufacturer (Rate nephelometry; IMMAGER, Beckman Coulter, USA). The measurement range is 0.1– 98 mg/dl. All samples from the same patient (batch samples) were measured in blinded pairs on the same ELISA kit to minimize run-to-run variability. The interassay and intraassay coefficients of variation were < 6%.
2.4. Statistical analysis Data are expressed as mean F S.D. or median (range, 25 – 75%). After testing data for normality, we used
K.K. Koh et al. / International Journal of Cardiology 95 (2004) 185–191 Table 1 Lipid effects of diet and simvastatin in hypercholesterolemic patients with coronary artery disease Variable
Diet + placebo
Diet + simvastatin
Baseline
Baseline
After diet
After therapy
Lipids (mg/dl) Total cholesterol 240 F 41 221 F 31* 226 F 33 163 F 37*,z Triglycerides 162 F 80 179 F 83 197 F 89 156 F 67*,z LDL cholesterol 153 F 27 134 F 36* 141 F 33 82 F 32*,z Apo B 118 F 26 110 F 24 110 F 18 81 F 17*,z HDL cholesterol 50 F 11 46 F 10* 46 F 10 48 F 11z Apo A-I 128 F 24 119 F 24* 116 F 23 123 F 20z LDL/HDL 3.25 F 0.86 3.02 F 0.89 3.23 F 1.06 1.76 F 0.63*,z cholesterol Apo B/apo 0.90 F 0.30 0.93 F 0.39 1.06 F 0.64 0.67 F 0.17*,z A-I ratio Data are expressed as mean F S.D. * P < 0.05 for comparison with the baseline value. z P < 0.05 for comparison with the value after therapy with diet.
Student’s paired t or Wilcoxon Signed Rank test to compare values between baseline and treatment for 14 weeks and Student’s unpaired t or Mann – Whitney Rank Sum test to compare baseline values and the percent changes between diet + placebo and diet + simvastatin for 14 weeks, as reported in Tables 1 and 2. Pearson or Spearman correlation coefficient analysis was used to assess associations between measured parameters. We calculated that 30 subjects would provide 80% power for detecting difference of absolute increase, 2.1% or greater flow-mediated dilation of the brachial artery between baseline and simvastatin, with a = 0.05 based on our previous studies [6]. A value of P < 0.05 was considered to be statistically significant.
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3. Results There were no significant differences in baseline characteristics and baseline values—lipids, vascular function (diameter and flow), and markers of inflammation levels— between diet + placebo and diet + simvastatin groups. 3.1. Effects of therapies on lipids The effects of therapies on lipids are shown in Table 1. Compared with diet alone, simvastatin significantly lowered total cholesterol and triglyceride levels ( P < 0.001 and P = 0.004, respectively), and lowered LDL cholesterol and apolipoprotein B levels ( P < 0.001 and P = 0.005, respectively), and increased HDL cholesterol and apolipoprotein A-I levels ( P = 0.017 and P = 0.040, respectively). Thus, the ratio of LDL to HDL cholesterol levels and apolipoprotein B to A-I significantly decreased with simvastatin (both P < 0.001) compared with diet alone. There were significant inverse correlations between pretreatment the ratio of LDL to HDL cholesterol or apolipoprotein B to A-I and the degree of change in those ratios after simvastatin (r = 0.409, P = 0.022 and r = 0.676, P < 0.001, respectively). 3.2. Effects of therapies on vasomotor function, nitrate, and malondialdehyde Both diet alone and simvastatin significantly improved the percent flow-mediated dilator response to hyperemia relative to pretreatment measurements (both P < 0.001), however, simvastatin significantly improved more than diet alone ( P < 0.001). The brachial artery dilator response to nitroglycerin between each therapy was not significantly
Table 2 Vascular effects of diet and simvastatin in hypercholesterolemic patients with coronary artery disease Variables
Diet + placebo
Diet + simvastatin
Baseline
After diet
Baseline
After therapy
Vasomotor function (%) Flow-mediated dilation Nitroglycerin dilation Nitrate (Amol/l) MDA (AM)
4.13 F 1.41 12.11 F 3.73 64 F 28 1.65 F 0.74
4.92 F 1.55* 12.68 F 3.49 69 F 34 1.41 F 0.55*
3.37 F 2.28 12.33 F 3.19 74 F 31 1.94 F 0.71
5.89 F 2.35*,z 12.59 F 3.36 63 F 34 1.58 F 0.75*
Cell adhesion molecules TNF-a (pg/ml) E-selectin (ng/ml) ICAM-1 (ng/ml) VCAM-1 (ng/ml)
2.59 (2.09 – 3.38) 41 (31 – 59) 229 (188 – 271) 425 (328 – 549)
2.81 (2.30 – 3.97)* 42 (30 – 63) 219 (186 – 270) 438 (310 – 557)
3.38 (1.24 – 4.45) 42 (33 – 56) 257 (196 – 290) 427 (331 – 552)
2.79 (1.20 – 3.87)*,z 41 (33 – 60) 225 (205 – 287)*,z 449 (313 – 571)
Acute-phase reactants C-reactive protein (mg/dl) Fibrinogen (mg/dl)
0.19 (0.12 – 0.48) 309 F 57
0.20 (0.11 – 0.51) 317 F 72
0.48 (0.15 – 1.23) 324 F 81
0.10 (0.10 – 0.26)*,z 282 F 84*,z
Data are expressed as mean F S.D. or median (25 – 75%). * P < 0.05 for comparison with the baseline value. z P < 0.05 for comparison with the value after therapy with diet.
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Fig. 1. Percent change in plasma levels of TNF-a from respective baseline levels after diet + placebo and diet + statin. Simvastatin significantly lowered plasma levels of TNF-a compared with diet alone. S.E.M. is identified by bars.
different ( P = 0.987; Table 2). Simvastatin lowered plasma levels of nitrate by 4 F 53% from the respective baseline levels ( P = 0.201) and significantly lowered plasma levels of MDA by 18 F 28% from the respective baseline levels ( P < 0.001). Compared with diet alone, simvastatin tended to lower plasma levels of nitrate and MDA (( P = 0.164 and P = 0.150, respectively, Table 2).
P = 0.014, respectively, Fig. 2). Further, we observed that patients with the highest baseline CRP levels showed the greatest extent of reductions on simvastatin (r = 0.582, P < 0.001). We investigated whether simvastatin-induced reduction in serological markers of inflammation was mediated by improvement in NO bioactivity or lipoprotein changes, compared with diet alone. There were significant inverse correlation between LDL cholesterol or the ratio of LDL to HDL cholesterol levels and flow-mediated dilation percent (r = 0.342, P = 0.009 and r = 0.356, P = 0.006, respectively). Of interest, there was significant inverse correlation between flow-mediated dilation percent and plasma levels of TNF-a (r = 0.329, P = 0.010; Fig. 3), however, no significant inverse correlation between flow-mediated dilation percent and CRP (r = 0.123, P = 0.365), E-selectin (r = 0.250, P = 0.183), ICAM-1 (r = 0.190, P = 0.314), or VCAM-1 levels (r = 0.067, P = 0.726). There was significant correlation between the changes in plasma nitrate levels and the changes in plasma levels of TNF-a (r = 0.497, P = 0.005). However, there was no significant inverse correlation between the changes in plasma nitrate levels and CRP (r = 0.071, P = 0.716), E-selectin (r = 0.260, P = 0.173), ICAM-1 (r = 0.029, P = 0.883), or VCAM-1 levels (r = 0.103, P = 0.596). Of more interest, no significant correlations between lipoprotein levels and levels of inflammation markers were determined ( 0.248 < r < 0.008). Despite the significant changes of lipoproteins, diet alone did not decrease the markers of inflammation. Furthermore, in order to identify a mechanism for the regulation of CRP and ICAM-1 levels, suggested by exper-
3.3. Effects of therapies on markers of inflammation 3.3.1. Cytokines and cell adhesion molecules Simvastatin significantly lowered plasma levels of TNF-a by 14 F 22% from the respective baseline levels ( P < 0.001) with more reduction than diet alone ( P < 0.001; Fig. 1). Further, we observed that patients with the highest baseline TNF-a levels showed the greatest extent of reductions on simvastatin (r = 0.442, P = 0.011). Simvastatin significantly decreased ICAM-1 levels by 8 F 18% from baseline values ( P = 0.041) with more reduction than diet alone ( P = 0.035). Further, we observed that patients with the highest baseline ICAM-1 levels showed the greatest extent of reductions on simvastatin (r = 0.437, P = 0.014). However, simvastatin did not significantly change plasma levels of E-selectin from 42 to 41 ( P = 0.364) and VCAM-1 from 427 to 449 ( P = 0.286). 3.3.2. Acute phase reactants Simvastatin significantly lowered serum levels of CRP and plasma levels of fibrinogen by 46 F 44% and 12 F 28%, respectively ( P < 0.001 and P = 0.022, respectively), with more reduction than diet alone ( P < 0.001 and
Fig. 2. Percent change in plasma levels of CRP from respective baseline levels after diet + placebo and diet + statin. Simvastatin significantly lowered serum levels of CRP compared with diet alone. S.E.M. is identified by bars.
K.K. Koh et al. / International Journal of Cardiology 95 (2004) 185–191
Fig. 3. Scatter plots showing the significant inverse correlation between flow-mediated dilation percent and plasma levels of TNF-a levels. The line shows the predicted regression line.
imental studies [14], we assessed correlations between CRP levels and ICAM-1 levels. There were no significant correlations between CRP levels and ICAM-1 levels ( 0.074 < r < 0.024).
4. Discussion We observed simvastatin significantly improved the percent flow-mediated dilator response to hyperemia and reduced levels of oxidant stress and inflammatory markers relative to baseline levels in hypercholesterolemic patients with coronary artery disease independent of lipoprotein changes, compared with diet alone. Several basic studies have confirmed that statins upregulate endothelial NO synthase and increase NO production [15-1,15-2]. We reasoned that simvastatin might improve endothelium-dependent vasomotor responsiveness by reducing oxidant stress and augmenting NO bioactivity. Therefore, we measured plasma nitrate and MDA levels. Although we did not measure the most sensitive marker of oxidant stress, prostaglandin F 2a, recent two studies reported a very good correlations between prostaglandin F2a and MDA levels [16,17]. Plasma nitrate levels, which reflect in part the luminal release of NO [18], were marginally reduced with simvastatin, which were consistent with our previous studies [6,19]. Reduction in luminal release of NO after simvastatin may indicate reduced synthesis of NO by constitutive NO synthase required for endothelial vasodialtor homeostasis as a consequence of reduced NO degradation by oxidized lipoproteins and free radical molecules from the
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endothelium and from inflammatory cells [20]. With reduced degradation of NO, NO synthesis may be decreased due to negative feedback effects of increased cytosolic levels of NO on NO synthase activity [20]. Simvastatin may also reduce the synthesis of NO from sources other than constitutive NO synthase. Increased expression of the inducible form of NO synthase in macrophages as well as normal cell lines within the vessel wall, capable of synthesizing even greater quantities of NO than the constitutive form of this enzyme, has been detected in human atherosclerotic plaques [21]. However, the reduction in plasma nitrate levels in our patients on therapies was not associated with changes in levels of markers of inflammation except TNF-a levels. In order to gain insight as to mechanisms of potential vasculoprotective effects of simvastatin, we measured surrogate markers for vascular inflammation. Inflammatory cytokines secreted by macrophages and T lymphocytes can modify endothelial function, smooth muscle cell proliferation, collagen degradation, and thrombosis [1,13]. We observed that simvastatin therapy significantly reduced plasma levels of TNF-a, ICAM-1, and serum levels of CRP, independently of lipoprotein changes. In the current study, simvastatin therapy had effects on LDL and HDL cholesterol levels, LDL/HDL cholesterol and its apolipoprotein ratios comparable to those achieved from our previous one [6]. Other mechanisms of potential benefit include improvement in vasomotor function and anti-inflammation. Cellular interaction between monocytes and endothelial cells was inhibited by statins, mediated via reducing the expression of adhesion molecules [22]. Of interest, the inhibitory effects of statins on the expression of adhesion molecules were completely reversed by the addition of mevalonate. We did observe that simvastatin reduced ICAM-1 levels, independent of changes in lipoprotein levels from pretreatment, further supporting non-lipid effects of statins [23,24]. TNF-a is a multifunctional circulating cytokine derived from endothelial and smooth muscle cells as well as macrophages associated with coronary atheroma [25]. Further, TNF-a enhances the rate of monocyte recruitment into developing atherosclerotic lesions. Ridker et al. [26] observed that plasma levels of TNF-a were persistently elevated among post-myocardial infarct patients at increased risk for recurrent coronary events. Of interest, the inhibitory effects of statins on the expression of TNF-a were completely reversed by the addition of mevalonate [27]. We did observe that simvastatin reduced plasma levels of TNF-a, independent of changes in lipoprotein levels from pretreatment, further supporting non-lipid effects of statins. ICAM-1 plays a pivotal role in leukocyte – endothelium interaction during the early stages of hypercholesterolemia in the rabbit [28] and indeed, ICAM-1 deficiency reduced atherosclerotic lesions in double-knockout mice (ApoE / /ICAM-1 / ) [29]. We did observe that simvas-
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tatin reduced ICAM-1 levels, independent of changes in lipoprotein levels from pretreatment, further supporting non-lipid effects of statins. CRP is a marker of inflammation [30]. Jialal et al. [4] demonstrated that simvastatin 20 mg daily significantly reduced CRP levels, not interleukin-6 levels and no relationship between reductions in CRP and LDL cholesterol in 22 combined hyperlipidemic patients, who received Step I Diet for 6 weeks and then used simvastatin for 6 weeks. Our 32 and 31 subjects were hyperlipidemic with established coronary artery disease and we compared the effects of simvastatin versus diet alone based on respective baseline levels. They did not measure flow-mediated vasodilation representing an important vascular effects of statins. We measured this in the same individuals. On the other hands, CRP is now considered to be important inflammatory mediator as well as acute phase reactant. They did not investigate the mechanism of CRP and other cytokines regulation as shown in the experimental studies [14]. In the same patients, we first investigated this and measured a bunch of other inflammatory markers important in the pathogenesis of atherosclerosis. We observed no correlations between CRP levels and lipoproteins or ICAM-1 levels in humans. Plasma levels of inflammatory markers were increased and correlated with the extent of disease in patients with atherosclerosis of the coronary and peripheral arteries [3]. Hingorani et al. [31] demonstrated that acute systemic inflammation with S. typhi vaccine impaired endotheliumdependent dilation in humans. Of interest, Raza et al. [32] reported flow-mediated dilation was significantly impaired in adults with primary systemic necrotizing vasculitis. Further, suppression of inflammation restored and normalized impaired endothelial function in these patients. We observed significant inverse correlations between TNF-a levels and flow-mediated dilation percent. Gaeta et al. [33] reported the offspring of patients with premature myocardial infarction had lower flow-mediated dilation, compared with the control subjects. Further, an inverse association was found between flow-mediated dilation and carotid intima-media thickness, two markers of early atherosclerosis. TNF-a, ICAM-1 and CRP are independent serological markers in predicting cardiovascular events. Further, of importance, we observed that patients with the highest pretreatment TNF-a, ICAM-1 and CRP levels showed the greatest extent of reductions on simvastatin. That is to say, patients with the highest pretreatment levels of inflammatory markers get the most benefits on simvastatin. This has a very important clinical implication in patients with established coronary artery disease because statin therapy was preferable and more efficacious than diet therapy. Our current observations are consistent with the Adult Treatment Panel III Guidelines reported in 2001 [34]. Taken together our current observations, increase of NO bioactivity and reduction of oxidant stress and inflammatory markers with simvastatin may contribute to the cardiovas-
cular event reduction and explain the early clinical benefit in clinical trials.
Acknowledgements We greatly appreciate for their critical review and comments regarding the manuscript to Richard O. Cannon III, M.D. (Clinical Director, National Heart, Lung, and Blood Institute, Bethesda, MD, USA) and Myron A. Waclawiw, Ph.D. (Office of Biostatistics Research, National Heart, Lung, and Blood Institute, Rockville, MD, USA).
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of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol 2001;21: 115 – 21. Wilson SH, Simari RD, Best PJM, et al. Simvastatin preserves coronary endothelial function in hypercholesterolemia in the absence of lipid-lowering. Arterioscler Thromb Vasc Biol 2001;21:122 – 8. Barath P, Fishbein MC, Cao J, et al. Detection and localization of tumor necrosis factor in human atheroma. Am J Cardiol 1990;65: 297 – 302. Ridker PM, et al, for the Cholesterol And Recurrent Events (CARE) Investigators. Elevation of tumor necrosis factor-a and increased risk of recurrent coronary events after myocardial infarction. Circulation 2000;101: 2149 – 2153. Pahan K, Sheikh FG, Namboodiri AM, Singh I. Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J Clin Invest 1997;100:2671 – 9. Scalia R, Appel III JZ, Lefer AM. Leukocyte-endothelium interaction during the early stages of hypercholesterolemia in the rabbit. Role of P-selectin, ICAM-1, and VCAM-1. Arterioscler Thromb Vasc Biol 1998;18:1093 – 100. Bourdillon M-C, Poston RN, Covacho C, et al. ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE / / ICAM-1 / ) fed a fat or a chow diet. Arterioscler Thromb Vasc Biol 2000;20:2630 – 5. Ridker PM, Hennekens CH, Buring JE, et al. C-reactive protein and other markers of inflammamtion in the prediction of cardiovascular disease in women. New Engl J Med 2000;342:836 – 43. Hingorani AD, Cross J, Kharbanda RK, et al. Acute systemic inflammation impairs endothelium-dependent dilatation in humans. Circulation 2000;102:994 – 9. Raza K, Thambyrajah J, Townend JN, et al. Suppression of inflammation in primary systemic vasculitis restores vascular endothelial function: lessons for atherosclerotic disease? Circulation 2000;102: 1470 – 2. Gaeta G, De Michele M, Cuomo S, et al. Arterial abnormalities in the offspring of patients with premature myocardial infarction. New Engl J Med 2000;343:840 – 6. Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III), J Am Med Assoc 2001;285:2486 – 97.
Vascular effects of diet and statin in hypercholesterolemic patients
$
Kwang Kon Koh a,*, Ji Won Son a, Jeong Yeal Ahn b, Dong Kyu Jin a, Hyung Sik Kim c, Yu Mi Choi d, Tae Hoon Ahn a, Dae Sung Kim e, Eak Kyun Shin a a
Vascular Medicine and Atherosclerosis Unit, Division of Cardiology, Gil Heart Center, Gachon Medical School, 1198 Kuwol-dong, Namdong-gu, Incheon 405-760, South Korea b Clinical Pathology, Gachon Medical School, Namdong-gu, Incheon, South Korea c Radiology, Gachon Medical School, Namdong-gu, Incheon, South Korea d Nutrition, Gachon Medical School, Namdong-gu, Incheon, South Korea e Preventive Medicine (Biostatistics), Gachon Medical School, Namdong-gu, Incheon, South Korea Received 26 November 2002; received in revised form 5 May 2003; accepted 6 May 2003
Abstract Objective: We investigated whether statin improves nitric oxide (NO) bioactivity and reduces serological markers of oxidant stress and inflammation and whether statin-induced reduction in markers of oxidant stress and inflammation is mediated by improvement in NO bioactivity or lipoprotein changes, compared with American Heart Association Step I Diet (Diet). Methods: We administered diet + placebo and diet + simvastatin 20 mg daily during 14 weeks with randomized order to 31 and 32 patients with coronary artery disease, respectively, with a randomized design. Results: Compared with diet alone, simvastatin significantly improved the percent flow-mediated dilator response to hyperemia and lowered plasma levels of tumor necrosis factor (TNF)-a, intercellular adhesion molecule type-1 (ICAM-1), serum levels of CRP, and fibrinogen ( P < 0.001, P < 0.001, P = 0.035, P < 0.001 and P = 0.014, respectively). Compared with diet alone, simvastatin lowered but statistically insignificant plasma levels of nitrate and malondialdehyde (MDA) ( P = 0.164 and P = 0.150, respectively). Further, we observed that patients with the highest pretreatment TNF-a, ICAM-1, and CRP levels showed the greatest extent of reductions on simvastatin. There were significant inverse correlation between low-density lipoprotein (LDL) cholesterol or the ratio of LDL to HDL cholesterol levels and flow-mediated dilation percent (r = 0.342, P = 0.009 and r = 0.356, P = 0.006, respectively). Of interest, there were significant inverse correlations between flow-mediated dilation percent and TNF-a levels (r = 0.329, P = 0.010). However, no significant correlations between lipoprotein levels and levels of inflammation markers were determined. Despite the significant changes of lipoproteins, diet alone did not decrease the markers of inflammation. Conclusions: Compared with diet alone, simvastatin significantly reduced markers of inflammation more. These effects were independent of lipoprotein changes. D 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: HMG-CoA reductase inhibitor; Diet; Endothelial function; Oxidant stress; Inflammation; Atherosclerosis
1. Introduction Vascular inflammation plays an important role in the pathogenesis of atherosclerosis [1]. Inflammation of the cap and shoulder of the plaque is a common feature in atherosclerotic femoral and coronary arteries [2]. Indeed, increased plasma levels of inflammatory markers were correlated with the extent of disease [3]. Statins reduce levels of some $ We presented our study in part as abstracts in the American Heart Association 75th Annual Scientific Session in Chicago in November 17 – 20, 2002. * Corresponding author. Tel.: +82-32-460-3683; fax: +82-32-460-3117/ 467-9302. E-mail address: [email protected] (K.K. Koh).
0167-5273/$ - see front matter D 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2003.05.018
markers of inflammation (e.g. C-reactive protein, CRP) when administered to hypercholesterolemic subjects, in a manner that appears to be independent of lipid-lowering effects [4]. Endothelial dysfunction of epicardial coronary arteries precedes development of atherosclerotic disease that is either angiographically apparent or of sufficient obstructive severity to cause myocardial ischemia and angina pectoris [5]. Patients with coronary heart disease or risk factors for coronary heart disease have been associated with impaired functions of the endothelium. The vessel wall in these conditions may promote inflammation, which contributes to development and clinical expression of atherosclerosis [1]. Statins improve endothelial function, consistent with enhanced nitric oxide (NO) bioactivity [6– 8].
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NO may protect nuclear transcription factor, NFnB, from activation by oxidized low-density lipoprotein (LDL) or cytokines, and thus prevent or attenuate the transcription and expression of adhesion molecules [9]. Thus, therapies that increase NO bioactivity may reduce synthesis of proinflammatory proteins on the endothelial cell surface, which may reduce inflammation. In this regard, we have previously shown that unopposed conjugated equine estrogen improved endothelium-dependent vasodilator responsiveness, an index of NO bioactivity and reduced adhesion molecules in healthy or hypercholesterolemic post-menopausal women [6,10]. Statins improved NO bioactivity in humans [6– 8] and reduced inflammatory cells within atherosclerotic plaque in experimental models [11]. However, recent studies suggest that the beneficial effects of statins on clinical events may involve non-lipid mechanisms that affect endothelial function: inflammatory responses, thrombus formation, and plaque stability [1,12,13]. The purpose of this study is to determine: (1) whether statin reduces serological markers of oxidant stress and inflammation potentially affected by NO-potentiating properties, (2) whether statin-induced reduction in markers of inflammation is mediated by improvement in NO bioactivity or lipoprotein changes, compared with Step I Diet, (3) the mechanism of CRP and other cytokines regulation because CRP induces the synthesis of cell adhesion molecules in monocytes and endothelial cells [14].
and h-blocker therapy on a long-term basis. The study was approved by the Gil Hospital Institute Review Board and all participants gave written, informed consent.
2. Methods
2.3. Vascular studies
2.1. Study population and design
Imaging studies of the right brachial artery were performed using a ATL HDI 3000 ultrasound machine equipped with a 10 MHz linear-array transducer, based on a previously published technique [6,10]. All images were transmitted to a personal computer via Ethernet with Digital Imaging and Communication in Medicine (DICOM) format and then saved on the hard disk of personal computer as a BMP format. Arterial diameters were measured with Image ToolR for Windows version 2.0 (University of Texas Health Science Center, San Antonio, TX). Measurements were performed by two independent investigators (D.K.J. and H.S.K.) blinded to the subject’s identity and medication status. Measurements of maximum diameter and percent flow-mediated dilation were made in 10 studies selected at random. The interobserver and intraobserver variability for repeated measurement of maximum diameter were 0.004 F 0.039 and 0.005 F 0.089 mm, respectively. The interobserver and intraobserver variability for repeated measurement of percent flow-mediated dilation were 0.07 F 1.27% and 0.15 F 1.24%, respectively.
Sixty-three patients with angiographically-documented coronary artery disease were enrolled in this study. We excluded patients with acute myocardial infarction or unstable angina in order to avoid the effects of acute ischemic injury on inflammatory process. All patients were in Canadian Cardiovascular Society class I or II. All patients were taught and placed on American Heart Association Step I Diet [14-1] through the study period. We administered diet + placebo and diet + simvastatin 20 mg daily during 14 weeks with randomized order to 31 and 32 patients with coronary artery disease, respectively, with a randomized design. Both groups were age- and sex-matched. The mean age of patients were 62-year-old and the number of male were 13 among 32. The risk factors were hypertension (68% vs. 72%; diet + placebo vs. diet + simvastatin), diabetes (23% vs. 25%), and current smoking (35% vs. 31%). The proportion of medications were h adrenergic blockers (81% vs. 81%), calcium channel blockers (48% vs. 47%), angiotensin converting enzyme inhibitors (23% vs. 28%), long-acting nitrates (81% vs. 84%), and aspirin (84% vs. 88%). Vasoactive medications, including calcium channel blockers, angiotensin converting enzyme inhibitors, and long-acting nitrates, were withheld for z 24 h before the study. All patients were taking aspirin
2.2. Laboratory assays Blood samples for laboratory assays were obtained at approximately 08:00 h. following overnight fasting before and after treatment for 14 weeks and immediately coded so that investigators performing laboratory assays were blinded to subject identity or study sequence. Assays for lipids, plasma nitrate (using the Griess reaction), malondialdehyde (MDA), E-selectin, intercellular adhesion molecule type-1 (ICAM-1), vascular cell adhesion molecule type-1 (VCAM1), and tumor necrosis factor (TNF)-a were performed in duplicate by ELISA (R&D Systems and BIOXYTECHR LPO-586, OxisResearch) as previously described [6,10,15]. Fibrinogen was measured by the thrombin-time titration method (Thrombocheck FibR, Japan). In all patients, serum was collected for the measurement of CRP levels. CRP levels were determined with an immunonephelometry system according to methods described by the manufacturer (Rate nephelometry; IMMAGER, Beckman Coulter, USA). The measurement range is 0.1– 98 mg/dl. All samples from the same patient (batch samples) were measured in blinded pairs on the same ELISA kit to minimize run-to-run variability. The interassay and intraassay coefficients of variation were < 6%.
2.4. Statistical analysis Data are expressed as mean F S.D. or median (range, 25 – 75%). After testing data for normality, we used
K.K. Koh et al. / International Journal of Cardiology 95 (2004) 185–191 Table 1 Lipid effects of diet and simvastatin in hypercholesterolemic patients with coronary artery disease Variable
Diet + placebo
Diet + simvastatin
Baseline
Baseline
After diet
After therapy
Lipids (mg/dl) Total cholesterol 240 F 41 221 F 31* 226 F 33 163 F 37*,z Triglycerides 162 F 80 179 F 83 197 F 89 156 F 67*,z LDL cholesterol 153 F 27 134 F 36* 141 F 33 82 F 32*,z Apo B 118 F 26 110 F 24 110 F 18 81 F 17*,z HDL cholesterol 50 F 11 46 F 10* 46 F 10 48 F 11z Apo A-I 128 F 24 119 F 24* 116 F 23 123 F 20z LDL/HDL 3.25 F 0.86 3.02 F 0.89 3.23 F 1.06 1.76 F 0.63*,z cholesterol Apo B/apo 0.90 F 0.30 0.93 F 0.39 1.06 F 0.64 0.67 F 0.17*,z A-I ratio Data are expressed as mean F S.D. * P < 0.05 for comparison with the baseline value. z P < 0.05 for comparison with the value after therapy with diet.
Student’s paired t or Wilcoxon Signed Rank test to compare values between baseline and treatment for 14 weeks and Student’s unpaired t or Mann – Whitney Rank Sum test to compare baseline values and the percent changes between diet + placebo and diet + simvastatin for 14 weeks, as reported in Tables 1 and 2. Pearson or Spearman correlation coefficient analysis was used to assess associations between measured parameters. We calculated that 30 subjects would provide 80% power for detecting difference of absolute increase, 2.1% or greater flow-mediated dilation of the brachial artery between baseline and simvastatin, with a = 0.05 based on our previous studies [6]. A value of P < 0.05 was considered to be statistically significant.
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3. Results There were no significant differences in baseline characteristics and baseline values—lipids, vascular function (diameter and flow), and markers of inflammation levels— between diet + placebo and diet + simvastatin groups. 3.1. Effects of therapies on lipids The effects of therapies on lipids are shown in Table 1. Compared with diet alone, simvastatin significantly lowered total cholesterol and triglyceride levels ( P < 0.001 and P = 0.004, respectively), and lowered LDL cholesterol and apolipoprotein B levels ( P < 0.001 and P = 0.005, respectively), and increased HDL cholesterol and apolipoprotein A-I levels ( P = 0.017 and P = 0.040, respectively). Thus, the ratio of LDL to HDL cholesterol levels and apolipoprotein B to A-I significantly decreased with simvastatin (both P < 0.001) compared with diet alone. There were significant inverse correlations between pretreatment the ratio of LDL to HDL cholesterol or apolipoprotein B to A-I and the degree of change in those ratios after simvastatin (r = 0.409, P = 0.022 and r = 0.676, P < 0.001, respectively). 3.2. Effects of therapies on vasomotor function, nitrate, and malondialdehyde Both diet alone and simvastatin significantly improved the percent flow-mediated dilator response to hyperemia relative to pretreatment measurements (both P < 0.001), however, simvastatin significantly improved more than diet alone ( P < 0.001). The brachial artery dilator response to nitroglycerin between each therapy was not significantly
Table 2 Vascular effects of diet and simvastatin in hypercholesterolemic patients with coronary artery disease Variables
Diet + placebo
Diet + simvastatin
Baseline
After diet
Baseline
After therapy
Vasomotor function (%) Flow-mediated dilation Nitroglycerin dilation Nitrate (Amol/l) MDA (AM)
4.13 F 1.41 12.11 F 3.73 64 F 28 1.65 F 0.74
4.92 F 1.55* 12.68 F 3.49 69 F 34 1.41 F 0.55*
3.37 F 2.28 12.33 F 3.19 74 F 31 1.94 F 0.71
5.89 F 2.35*,z 12.59 F 3.36 63 F 34 1.58 F 0.75*
Cell adhesion molecules TNF-a (pg/ml) E-selectin (ng/ml) ICAM-1 (ng/ml) VCAM-1 (ng/ml)
2.59 (2.09 – 3.38) 41 (31 – 59) 229 (188 – 271) 425 (328 – 549)
2.81 (2.30 – 3.97)* 42 (30 – 63) 219 (186 – 270) 438 (310 – 557)
3.38 (1.24 – 4.45) 42 (33 – 56) 257 (196 – 290) 427 (331 – 552)
2.79 (1.20 – 3.87)*,z 41 (33 – 60) 225 (205 – 287)*,z 449 (313 – 571)
Acute-phase reactants C-reactive protein (mg/dl) Fibrinogen (mg/dl)
0.19 (0.12 – 0.48) 309 F 57
0.20 (0.11 – 0.51) 317 F 72
0.48 (0.15 – 1.23) 324 F 81
0.10 (0.10 – 0.26)*,z 282 F 84*,z
Data are expressed as mean F S.D. or median (25 – 75%). * P < 0.05 for comparison with the baseline value. z P < 0.05 for comparison with the value after therapy with diet.
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Fig. 1. Percent change in plasma levels of TNF-a from respective baseline levels after diet + placebo and diet + statin. Simvastatin significantly lowered plasma levels of TNF-a compared with diet alone. S.E.M. is identified by bars.
different ( P = 0.987; Table 2). Simvastatin lowered plasma levels of nitrate by 4 F 53% from the respective baseline levels ( P = 0.201) and significantly lowered plasma levels of MDA by 18 F 28% from the respective baseline levels ( P < 0.001). Compared with diet alone, simvastatin tended to lower plasma levels of nitrate and MDA (( P = 0.164 and P = 0.150, respectively, Table 2).
P = 0.014, respectively, Fig. 2). Further, we observed that patients with the highest baseline CRP levels showed the greatest extent of reductions on simvastatin (r = 0.582, P < 0.001). We investigated whether simvastatin-induced reduction in serological markers of inflammation was mediated by improvement in NO bioactivity or lipoprotein changes, compared with diet alone. There were significant inverse correlation between LDL cholesterol or the ratio of LDL to HDL cholesterol levels and flow-mediated dilation percent (r = 0.342, P = 0.009 and r = 0.356, P = 0.006, respectively). Of interest, there was significant inverse correlation between flow-mediated dilation percent and plasma levels of TNF-a (r = 0.329, P = 0.010; Fig. 3), however, no significant inverse correlation between flow-mediated dilation percent and CRP (r = 0.123, P = 0.365), E-selectin (r = 0.250, P = 0.183), ICAM-1 (r = 0.190, P = 0.314), or VCAM-1 levels (r = 0.067, P = 0.726). There was significant correlation between the changes in plasma nitrate levels and the changes in plasma levels of TNF-a (r = 0.497, P = 0.005). However, there was no significant inverse correlation between the changes in plasma nitrate levels and CRP (r = 0.071, P = 0.716), E-selectin (r = 0.260, P = 0.173), ICAM-1 (r = 0.029, P = 0.883), or VCAM-1 levels (r = 0.103, P = 0.596). Of more interest, no significant correlations between lipoprotein levels and levels of inflammation markers were determined ( 0.248 < r < 0.008). Despite the significant changes of lipoproteins, diet alone did not decrease the markers of inflammation. Furthermore, in order to identify a mechanism for the regulation of CRP and ICAM-1 levels, suggested by exper-
3.3. Effects of therapies on markers of inflammation 3.3.1. Cytokines and cell adhesion molecules Simvastatin significantly lowered plasma levels of TNF-a by 14 F 22% from the respective baseline levels ( P < 0.001) with more reduction than diet alone ( P < 0.001; Fig. 1). Further, we observed that patients with the highest baseline TNF-a levels showed the greatest extent of reductions on simvastatin (r = 0.442, P = 0.011). Simvastatin significantly decreased ICAM-1 levels by 8 F 18% from baseline values ( P = 0.041) with more reduction than diet alone ( P = 0.035). Further, we observed that patients with the highest baseline ICAM-1 levels showed the greatest extent of reductions on simvastatin (r = 0.437, P = 0.014). However, simvastatin did not significantly change plasma levels of E-selectin from 42 to 41 ( P = 0.364) and VCAM-1 from 427 to 449 ( P = 0.286). 3.3.2. Acute phase reactants Simvastatin significantly lowered serum levels of CRP and plasma levels of fibrinogen by 46 F 44% and 12 F 28%, respectively ( P < 0.001 and P = 0.022, respectively), with more reduction than diet alone ( P < 0.001 and
Fig. 2. Percent change in plasma levels of CRP from respective baseline levels after diet + placebo and diet + statin. Simvastatin significantly lowered serum levels of CRP compared with diet alone. S.E.M. is identified by bars.
K.K. Koh et al. / International Journal of Cardiology 95 (2004) 185–191
Fig. 3. Scatter plots showing the significant inverse correlation between flow-mediated dilation percent and plasma levels of TNF-a levels. The line shows the predicted regression line.
imental studies [14], we assessed correlations between CRP levels and ICAM-1 levels. There were no significant correlations between CRP levels and ICAM-1 levels ( 0.074 < r < 0.024).
4. Discussion We observed simvastatin significantly improved the percent flow-mediated dilator response to hyperemia and reduced levels of oxidant stress and inflammatory markers relative to baseline levels in hypercholesterolemic patients with coronary artery disease independent of lipoprotein changes, compared with diet alone. Several basic studies have confirmed that statins upregulate endothelial NO synthase and increase NO production [15-1,15-2]. We reasoned that simvastatin might improve endothelium-dependent vasomotor responsiveness by reducing oxidant stress and augmenting NO bioactivity. Therefore, we measured plasma nitrate and MDA levels. Although we did not measure the most sensitive marker of oxidant stress, prostaglandin F 2a, recent two studies reported a very good correlations between prostaglandin F2a and MDA levels [16,17]. Plasma nitrate levels, which reflect in part the luminal release of NO [18], were marginally reduced with simvastatin, which were consistent with our previous studies [6,19]. Reduction in luminal release of NO after simvastatin may indicate reduced synthesis of NO by constitutive NO synthase required for endothelial vasodialtor homeostasis as a consequence of reduced NO degradation by oxidized lipoproteins and free radical molecules from the
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endothelium and from inflammatory cells [20]. With reduced degradation of NO, NO synthesis may be decreased due to negative feedback effects of increased cytosolic levels of NO on NO synthase activity [20]. Simvastatin may also reduce the synthesis of NO from sources other than constitutive NO synthase. Increased expression of the inducible form of NO synthase in macrophages as well as normal cell lines within the vessel wall, capable of synthesizing even greater quantities of NO than the constitutive form of this enzyme, has been detected in human atherosclerotic plaques [21]. However, the reduction in plasma nitrate levels in our patients on therapies was not associated with changes in levels of markers of inflammation except TNF-a levels. In order to gain insight as to mechanisms of potential vasculoprotective effects of simvastatin, we measured surrogate markers for vascular inflammation. Inflammatory cytokines secreted by macrophages and T lymphocytes can modify endothelial function, smooth muscle cell proliferation, collagen degradation, and thrombosis [1,13]. We observed that simvastatin therapy significantly reduced plasma levels of TNF-a, ICAM-1, and serum levels of CRP, independently of lipoprotein changes. In the current study, simvastatin therapy had effects on LDL and HDL cholesterol levels, LDL/HDL cholesterol and its apolipoprotein ratios comparable to those achieved from our previous one [6]. Other mechanisms of potential benefit include improvement in vasomotor function and anti-inflammation. Cellular interaction between monocytes and endothelial cells was inhibited by statins, mediated via reducing the expression of adhesion molecules [22]. Of interest, the inhibitory effects of statins on the expression of adhesion molecules were completely reversed by the addition of mevalonate. We did observe that simvastatin reduced ICAM-1 levels, independent of changes in lipoprotein levels from pretreatment, further supporting non-lipid effects of statins [23,24]. TNF-a is a multifunctional circulating cytokine derived from endothelial and smooth muscle cells as well as macrophages associated with coronary atheroma [25]. Further, TNF-a enhances the rate of monocyte recruitment into developing atherosclerotic lesions. Ridker et al. [26] observed that plasma levels of TNF-a were persistently elevated among post-myocardial infarct patients at increased risk for recurrent coronary events. Of interest, the inhibitory effects of statins on the expression of TNF-a were completely reversed by the addition of mevalonate [27]. We did observe that simvastatin reduced plasma levels of TNF-a, independent of changes in lipoprotein levels from pretreatment, further supporting non-lipid effects of statins. ICAM-1 plays a pivotal role in leukocyte – endothelium interaction during the early stages of hypercholesterolemia in the rabbit [28] and indeed, ICAM-1 deficiency reduced atherosclerotic lesions in double-knockout mice (ApoE / /ICAM-1 / ) [29]. We did observe that simvas-
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tatin reduced ICAM-1 levels, independent of changes in lipoprotein levels from pretreatment, further supporting non-lipid effects of statins. CRP is a marker of inflammation [30]. Jialal et al. [4] demonstrated that simvastatin 20 mg daily significantly reduced CRP levels, not interleukin-6 levels and no relationship between reductions in CRP and LDL cholesterol in 22 combined hyperlipidemic patients, who received Step I Diet for 6 weeks and then used simvastatin for 6 weeks. Our 32 and 31 subjects were hyperlipidemic with established coronary artery disease and we compared the effects of simvastatin versus diet alone based on respective baseline levels. They did not measure flow-mediated vasodilation representing an important vascular effects of statins. We measured this in the same individuals. On the other hands, CRP is now considered to be important inflammatory mediator as well as acute phase reactant. They did not investigate the mechanism of CRP and other cytokines regulation as shown in the experimental studies [14]. In the same patients, we first investigated this and measured a bunch of other inflammatory markers important in the pathogenesis of atherosclerosis. We observed no correlations between CRP levels and lipoproteins or ICAM-1 levels in humans. Plasma levels of inflammatory markers were increased and correlated with the extent of disease in patients with atherosclerosis of the coronary and peripheral arteries [3]. Hingorani et al. [31] demonstrated that acute systemic inflammation with S. typhi vaccine impaired endotheliumdependent dilation in humans. Of interest, Raza et al. [32] reported flow-mediated dilation was significantly impaired in adults with primary systemic necrotizing vasculitis. Further, suppression of inflammation restored and normalized impaired endothelial function in these patients. We observed significant inverse correlations between TNF-a levels and flow-mediated dilation percent. Gaeta et al. [33] reported the offspring of patients with premature myocardial infarction had lower flow-mediated dilation, compared with the control subjects. Further, an inverse association was found between flow-mediated dilation and carotid intima-media thickness, two markers of early atherosclerosis. TNF-a, ICAM-1 and CRP are independent serological markers in predicting cardiovascular events. Further, of importance, we observed that patients with the highest pretreatment TNF-a, ICAM-1 and CRP levels showed the greatest extent of reductions on simvastatin. That is to say, patients with the highest pretreatment levels of inflammatory markers get the most benefits on simvastatin. This has a very important clinical implication in patients with established coronary artery disease because statin therapy was preferable and more efficacious than diet therapy. Our current observations are consistent with the Adult Treatment Panel III Guidelines reported in 2001 [34]. Taken together our current observations, increase of NO bioactivity and reduction of oxidant stress and inflammatory markers with simvastatin may contribute to the cardiovas-
cular event reduction and explain the early clinical benefit in clinical trials.
Acknowledgements We greatly appreciate for their critical review and comments regarding the manuscript to Richard O. Cannon III, M.D. (Clinical Director, National Heart, Lung, and Blood Institute, Bethesda, MD, USA) and Myron A. Waclawiw, Ph.D. (Office of Biostatistics Research, National Heart, Lung, and Blood Institute, Rockville, MD, USA).
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