- Email: [email protected]
Anti-inflammatory effects of simvastatin in subjects with hypercholesterolemia
International Journal of Cardiology 77 (2001) 247–253 www.elsevier.com / locate / ijcard
Anti-inflammatory effects of simvastatin in subjects with hypercholesterolemia Jacek Musial*, Anetta Undas, Piotr Gajewski, Milosz Jankowski, Wojciech Sydor, Andrzej Szczeklik Department of Medicine, Jagiellonian University School of Medicine, Skawinska 8, 31 -066 Krakow, Poland Received 19 July 2000; received in revised form 24 October 2000; accepted 28 October 2000
Abstract Aims: Beneficial effects of statins in preventing cardiovascular events may depend, at least in part, on their anti-inflammatory action. The aim of the study was to assess the influence of simvastatin and aspirin on serum levels of C-reactive protein (CRP), tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) in hypercholesterolemic subjects. Methods and results: In 33 asymptomatic men with total cholesterol (TC) .6.5 mmol l 21 and in 25 men with coronary heart disease and borderline-high cholesterol levels (between 5.2 and 6.5 mmol l 21 ) chronically treated with low-dose aspirin (75 mg / d), serum levels of CRP, TNF-a, IL-6, and IL-8 were determined before and after a 3-month simvastatin therapy (20–40 mg daily). In the former group, these markers of inflammation were also measured before and after a 2-week treatment with aspirin (300 mg / d), implemented prior to and in combination with simvastatin. A distinct reduction of CRP and TNF-a was found in both groups; IL-6 levels were decreased only in subjects with marked hypercholesterolemia. Aspirin had no effect on the anti-inflammatory action of simvastatin. Conclusions: In men with hypercholesterolemia simvastatin treatment lowers serum levels of CRP and proinflammatory cytokines. Low-dose aspirin does not add to the anti-inflammatory action of simvastatin. 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Simvastatin; Aspirin; Hypercholesterolemia; C-reactive protein; Proinflammatory cytokines
1. Introduction Atherosclerosis is now considered an inflammatory disease [1]. This particular type of arterial inflammation may be initiated and sustained by several factors. Elevated levels of plasma cholesterol, particularly low-density lipoprotein (LDL) cholesterol, are one of the most important risk factors for cardiovascular disease [2]. In the arterial wall LDL particles may be modified and taken up by macrophage scavenger
*Corresponding author. Tel.: 148-12-656-2840; fax: 148-12-6565786. E-mail address: [email protected] (J. Musial).
receptors. This leads to the foam cell formation and initiation of an inflammatory response [1,3]. C-reactive protein (CRP) is a major acute phase protein and a known clinical marker of inflammation. Its production in the liver remains under strict control of proinflammatory interleukin-1-type (e.g. tumor necrosis factor-a-TNF-a) and interleukin-6-type (e.g. interleukin-6–IL-6) cytokines [4]. Plasma concentration of CRP correlates with severity and extent of the atherosclerotic process [5] and may mediate itself the inflammatory process in the arterial wall [6]. Higher CRP levels could serve as a prognostic marker of coronary events in survivors of myocardial infarction and in patients with stable or unstable angina [7–9]. Moreover, CRP level predicts the first
0167-5273 / 01 / $ – see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 00 )00439-3
248
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
myocardial infarction and other atherothrombotic events in apparently healthy men [10–12]. Lowering of serum cholesterol with statins (3hydroxy-3-methylglutaryl-CoA reductase inhibitors), used in the primary and secondary prevention of coronary heart disease (CHD), reduces the risk of acute cardiovascular events and prolongs survival. Beneficial effects of statins extend across a wide range of serum cholesterol levels [13–15]. Recently, besides lipid-lowering action, nonlipid effects of statins, including antithrombotic [16] and anti-inflammatory [17] properties, have been strongly suggested [5,18]. Two preliminary, recent reports indicate that treatment with statins could lower plasma CRP levels in hyperlipidemic coronary patients [19], and decrease TNF-a and IL-6 concentrations in lipopolysaccharide (LPS)-stimulated blood samples [20]. Therefore, we decided to study in detail the effects of simvastatin treatment on plasma levels of CRP and proinflammatory cytokines (TNF-a and IL-6) in subjects with elevated serum total cholesterol (TC) concentrations. We also compared the effects of simvastatin versus that of aspirin. For this purpose, in a group of patients with marked hypercholesterolemia 21 (TC .6.5 mmol l ), we investigated the action of a 2-week treatment with aspirin before and after lowering cholesterol levels by simvastatin.
2. Methods The design of the study (Group 1) has been described in detail elsewhere [16]. In brief, the study enrolled 33 healthy male volunteers with a mean age of 47.4 years (range, 34 to 61 years) whose serum TC was .6.5 mmol l 21 , LDL cholesterol .3.6 mmol l 21 , and fasting triglycerides ,4.6 mmol l 21 during 6 weeks of treatment with the National Cholesterol Education Program Step 1 diet. Subjects were eligible if they have fasting plasma glucose level ,6.7 mmol l 21 , creatinine ,120 mmol l 21 , absence of symptomatic CHD or congestive heart failure or other severe chronic illness (including a history of venous thromboembolism). Subjects were not allowed to take aspirin or any agents interfering with hemostasis for at least 4 weeks before entering the study. Twenty
men smoked regularly (on average, 10 cigarettes daily). During the first 2 weeks of the study, subjects received 300 mg of aspirin daily (Phase I). Then, they were given simvastatin for 12 weeks (Phase II). The starting dose of the drug was 20 mg taken once at bedtime. The dose was doubled after 4 weeks, if the reduction in TC was less than 1 mmol l 21 . The maximal dose of simvastatin was 40 mg a day. Finally, aspirin 300 mg / d was added to simvastatin for the additional 2 weeks (Phase III). Haemostatic parameters and lipid profiles, analysed in this group, have already been published [16]. Group 2 consisted of 25 age-matched patients (range; 39 to 64 years) with borderline-high cholesterol levels (between 5.2 and 6.5 mmol l 21 ; median 5.91 mmol l 21 ). For inclusion, patients were required to have a history (at least 6 month before entering the study) of myocardial infarction or hospitalization due to unstable angina pectoris. Other inclusion and exclusion criteria were identical to those accepted for the subjects with marked hypercholesterolemia. Six men were current smokers (on average, 10 cigarettes daily). Patients were regularly treated with 75 mg of aspirin daily. Simvastatin, as a study agent, was given for 12 weeks at a dose of 20 mg a day. The therapeutic goal was to reduce TC below 5.2 mmol l 21 . At the study entry, none of the men enrolled had any clinical signs or symptoms of infection. Informed consent to participate in the study was obtained from all individuals. Fasting venous blood samples were collected with minimal stasis between 8 a.m. and 10 a.m. Serum samples for inflammatory markers were frozen at 2808C until assayed, but no longer than 12 months. Serum levels of CRP were measured nephelometrically (Behring, Marburg, Germany) using a sensitive assay (detection limit 0.16 mg l 21 of the protein). Reported intra-assay and inter-assay coefficients of variation were 4% and 2.6%, respectively. Concentrations of TNFa and IL-6 in serum were determined by chemiluminescent enzyme immunometric assays, using the IMMUNOLITE Automated Analyzer (DPC, Los Angeles, USA). Intra-assay and interassay coefficients of variation were 3.5% and 6.5%, and 7.9% and 3.6%, respectively. The detection limits of the assays were approximately 1.7 pg ml 21 for TNF-a and 1 pg ml 21 for IL-6). In subjects with
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
marked hypercholesterolemia all the markers of inflammation were determined 4 times. They were performed at baseline (week 0), after 2 weeks of aspirin administration (Phase I), after a 3-month simvastatin treatment (Phase II) and finally, after the 2 weeks of combined aspirin and simvastatin treatment (Phase III). In the Group 2 determinations were performed twice: at baseline and at the end of 12 week simvastatin treatment. All samples were coded in order to blind the laboratory personnel regarding subject’s name and phase of treatment.
3. Statistical analysis Since inflammatory variables in Group 1 were not normally distributed, repeated measure analysis was performed by Friedman’s test, followed by paired analysis using Wilcoxon matched pairs test. Correlations were assessed by Spearman rank order test. In Group 2 inflammatory variables also did not follow normal distribution and were compared by Wilcoxon matched pairs test. Values were expressed as median and interquartile range. Statistical significance was accepted at a level of P,0.05. Calculations were performed on a PC computer using Statistica (Statsoft Inc., Tulsa, OK, USA) software.
4. Results As previously reported [16], in men with marked hypercholesterolemia (Group 1), a 3-month simvastatin treatment led to a reduction of TC by 31% (from a median of 7.85–5.43 mmol l 21 ; P , 0.00001), LDL cholesterol by 41% (from a median of 5.57–3.26 mmol l 21 ; P , 0.00001), and triglycerides by 26%
249
(from a median of 2.32–1.72 mmol l 21 ; P , 0.00001), whereas HDL cholesterol was raised by 7.5% (from a median of 1.34–1.44 mmol l 21 ; P . 0.05). Aspirin did not influence serum CRP concentrations. Treatment with simvastatin for 3 months was associated with a significant reduction in CRP levels by about 50%. A similar fall (by about 60%) of CRP levels was found after the combined treatment with aspirin (300 mg / d) added for 2 weeks to simvastatin. However, the difference between Phase II and Phase III values did not reach the level of statistical significance (Table 1, Fig. 1A). In none of the subjects studied the level of CRP exceeded 10 mg l 21 . There was a significant correlation between CRP level at baseline and its relative change after simvastatin treatment (R50.70, P , 0.0001). It suggests a greater reduction in serum CRP levels by simvastatin in subjects with higher baseline values of this marker. Men with CRP.1 mg l 21 (n518) had a 12-fold higher chance of reduction in CRP levels after a 3-month simvastatin therapy (odds ratio [OR]5 12.00; 95% confidence interval [CI]: 2.23–64.65; P 5 0.003). After the combined treatment with aspirin and simvastatin this effect was even more pronounced (OR514.00; 95% CI: 1.86–104.85; P 5 0.008). Interestingly, serum TNF-a levels were decreased slightly, but significantly, after aspirin alone. Simvastatin treatment reduced TNFa levels by about 20% in comparison with the initial values. Addition of aspirin to simvastatin for the additional 2 weeks reduced TNFa values as compared to baseline and the levels measured after aspirin alone. This combination, however, did not significantly change TNFa levels, decreased previously by simvastatin treatment (Table 1, Fig. 1A). Aspirin had no effect on serum IL-6 levels, while
Table 1 Influence of aspirin and simvastatin on markers of inflammation in patients with marked hipercholesterolemia (Group 1)a Variables
At baseline
After aspirin (Phase I)
After simvastatin (Phase II)
After simvastatin1aspirin (Phase III)
P
C-reactive protein (mg l 21 ) TNF-a (pg?ml 21 ) Interleukin-6 (pg?ml 21 )
1.02 (0.44; 2.06) 4.9 (4.2; 7.9) 4.6 (2.7; 10.2)
1.04 (0.49; 1.51) 4.6 (4.0; 6.5)† 4.1 (3.0; 12.2)
0.52 (0.37; 1.35)* 4.0 (3.6; 4.3)† 2.7 (2.1; 3.4)*
0.39 (0.27; 0.89)†‡ 3.9 (3.6; 4.1)†§ 2.2 (2.0; 4.05)†‡
0.002 ,0.00001 0.0004
a
Values are median (quartile 1;3). Friedman’s test was applied followed by paired analysis using Wilcoxon test: *, P,0.05 as compared to baseline; †, P , 0.01 as compared to baseline; ‡, P , 0.01 as compared to phase I; §, P , 0.05 as compared to phase I.
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
250
Fig. 1. Concentrations (medians) of C-reactive protein (CRP), tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6) during simvastatin or / and aspirin treatment, expressed as a percentage of baseline values (100%). Bars denote interquartile ranges. a – changes in men with marked hypercholesterolemia – group 1 (n533) b – changes in men with borderline-high hypercholesterolemia – group 2 (n525).
simvastatin reduced it by about 40% as compared to the pretreatment values. After two additional weeks of combined aspirin and simvastatin administration IL-6 levels fell by more than 50%. There was no difference between Phase II and Phase III values (Table 1, Fig. 1A). Three-month simvastatin treatment of CHD patients with borderline high hypercholesterolemia (Group 2) led to the reduction of TC by about 25% (from a median of 5.91–4.39 mmol l 21 ; P , 0.0001), LDL-cholesterol by about 40% (from a median of 3.71–2.23 mmol l 21 ; P , 0.0001), while the levels of HDL-cholesterol and triglycerides remained unchanged. At the same time CRP levels decreased by more than 25% (Table 2) These changes were
accompanied by a slight but significant fall in the TNF-a levels, while serum IL-6 levels showed no change. There was no significant difference between Group 1 and Group 2 baseline levels of inflammatory markers studied, even if Group 2 was on chronic low-dose aspirin treatment. Interestingly, however, simvastatin had an almost four-fold greater chance to reduce TNF-a levels in subjects with marked hypercholesterolemia than in these with borderline-high TC values (OR53.6; 95% CI: 1.08–11.93). There were no similar relationships with regard to the remaining markers of inflammation. No correlation could be found between CRP, TNFa, and IL-6 values, before as well as after drug
Table 2 Influence of simvastatin on markers of inflammation in patients with moderate hipercholesterolemia (Group 2) under long-term aspirin treatment (75 mg / d)a Variables 21
C-reactive protein (mg l ) TNF-a (pg?ml 21 ) Interleukin-6 (pg?ml 21 ) a
At baseline
After simvastatin
P
1.61 (0.80; 2.88) 4.3 (4.0; 8.4) 2.9 (2.0; 16.3)
1.17 (0.31; 2.02) 4.0 (4.0; 4.5) 3.9 (2.0; 25.3)
0.011 0.015 N.S.
Values are median (quartile 1;3). Wilcoxon test was applied for statistical analysis.
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
interventions in either group. There was also no relation between lipid and inflammatory parameters, with an exception of a significant correlation between baseline TNF-a and TC levels in Group 1 (R 5 0.67; P , 0.0001). There was no difference in markers of inflammation depending on the smoking status in both groups. Change in serum levels of CRP, TNF-a, and IL-6 was not related to the dose of simvastatin given to the patients in Group 1; nine received 40 mg of the drug daily.
5. Discussion We have shown that in men with hypercholesterolemia a 3-month simvastatin treatment significantly reduces serum markers of inflammation. To our knowledge, no previous study has sought to explore changes in all the 3 markers: CRP, TNF-a and IL-6, measured in peripheral venous blood in subjects treated with statins. The significant reduction was particularly evident in subjects with markedly elevated cholesterol levels (.6.5 mmol l 21 ), in whom CRP, TNF-a, and IL-6, all decreased substantially. Less pronounced, but significant reductions in CRP and TNF-a were also observed in CHD patients with borderline high cholesterol-levels, who were already treated with low-dose aspirin. These results corroborate preliminary data, published by Strandberg et al. [19] and Rosenson et al. [20]. However, the former analysis was performed after 8 weeks of treatment with either atorvastatin or simvastatin given in the blinded fashion in a study, which is still underway. In the latter, reductions of TNF-a and IL-6 were observed in 7 subjects taking pravastatin, another HMG-CoA inhibitor, for 7 weeks but only when their blood was stimulated in vitro by lipopolysaccharide. No change in the IL-1b, IL-8 and IL-1 receptor antagonist was noted [20]. Recently, Ridker et al. [21] reported a reduction in plasma concentrations of CRP in participants of the CARE trial after 5 years of treatment with pravastatin. Beneficial effects of statins in atherosclerosis extend probably beyond their cholesterol-lowering action. Anti-inflammatory properties may be particularly important in protecting vessel wall and preventing acute vascular episodes. Evidence from ex-
251
perimental and human studies indicate that statins indeed possess such properties, which are likely to be independent from lipid-lowering effects [7,22]. Indeed, in a recent study Ridker et al. [21] could not show any correlation between lipid-lowering action of pravastatin and its ability to lower CRP levels. In experimental animals, pravastatin and atorvastatin reduce arterial inflammation [22,23]. On the other hand, lovastatin and simvastatin decrease monocyte CD11b surface expression and monocyte adhesion to endothelium in hypercholesterolemic patients [24]. In addition, fluvastatin could inhibit the secretion of matrix metalloproteinases from macrophages, and by this effect possibly prevent atherosclerotic plaque disruption [25]. The mechanism of anti-inflammatory action of statins has not been elucidated. Some evidence points to the decreased production of mevalonate and isoprenoids, the early products generated by HMG-CoA reductase [17,25]. The effect of simvastatin on CRP levels in marked hypercholesterolemia appears to depend on the initial levels of this protein. This significant correlation, found between the baseline concentrations of CRP and its statin-induced reduction, may be of clinical importance, particularly, in the light of the finding that patients with the highest serum levels of CRP prior to treatment with pravastatin had the highest risk for recurrent coronary events [7]. Further studies are needed to show whether clinical benefits from statin treatment are related to initial levels of markers of inflammation and degree of their reduction. Two-week treatment with 300 mg of aspirin per day did not influence the concentrations of serum inflammation markers, except for a slight fall in the TNF-a levels. No enhancement of anti-inflammatory effect was observed when aspirin was added for 2 weeks to simvastatin. This study was not designed to assess the effects of chronic aspirin treatment on serum markers of inflammation. For this reason we are unable to explain whether a less pronounced anti-inflammatory effect of simvastatin in patients with borderline-high cholesterol depends on an additional effect of low-dose aspirin treatment, or is a result of lower total cholesterol levels. While elder studies suggested that aspirin had no influence on CRP levels [26–28], a recent report showed 6 weeks of aspirin treatment in a daily
252
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
dose of 300 mg lowers plasma levels of CRP, IL-6 and macrophage colony stimulating factor in patients with coronary heart disease [29]. In cell culture aspirin is able to inhibit TNF-a release [30]. Interestingly, aspirin has been shown to be particularly effective in preventing the first myocardial infarction in men with the highest C-reactive protein values [11]. The limitation of the current study is the absence of placebo group. However, most of subjects from group 1 have at least 2 coronary risk factors, in addition to marked hypercholesterolemia, while those from group 2 have documented coronary artery disease, we considered it unethical to postpone cholesterol-lowering treatment.
6. Conclusions In subjects with a broad range of cholesterol levels simvastatin exerts a marked anti-inflammatory effect. This could explain, at least in part, be responsible for the clinical effectiveness of the drug in lowering the risk of acute cardiovascular complications and prolonging life. It has been suggested that anti-inflammatory agents may provide clinical benefits in preventing cardiovascular disease [11]. Simvastatin certainly belongs to such agents.
References [1] Ross R. Atherosclerosis – an inflammatory disease. New Engl J Med 1999;340:115–26. [2] National Cholesterol Education Program. Second report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). Circulation 1994;89:1329–445. [3] Steinberg D. Low-density lipoprotein oxidation and its pathobiological significance. J Biol Chem 1997;272:20963–6. [4] Baumann H, Gauldie J. The acute phase response. Immunol Today 1994;15:74–80. ¨ ¨ [5] Heinrich J, Schulte H, Schonfeld R, Kohler E, Assmann G. Association of variables of coagulation, fibrinolysis and acute-phase with atherosclerosis in coronary and peripheral arteries and those arteries supplying the brain. Thromb Haemost 1995;73:374–9. [6] Lagrand WK, Visser CA, Hermens WT et al. C-reactive protein as a cardiovascular risk factor. More then an epiphenomenon? Circulation 1999;100:96–102. [7] Ridker PM, Rifai N, Pfeffer MA et al., for the Cholesterol and Recurrent Events (CARE) Investigators. Inflammation, pravastatin and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Circulation 1998;98:839– 44.
[8] Haverkate F, Thompson SG, Pyke SD, Gallimore JR, Pepys MB, for the European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. Production of C-reactive protein and risk of coronary events in stable and unstable angina. Lancet 1997;349:462–6. [9] Verheggen PWHM, de Maat MPM, Manger Cats V et al. Inflammatory status as a main determinant of outcome in patients with unstable angina, independent of coagulation activation and endothelial cell function. Eur Heart J 1999;20:567–74. [10] Ridker PM, Glynn RJ, Hennekens CH. C-reactive protein adds to the predictive value of total and HDL cholesterol in determining risk of first myocardial infarction. Circulation 1998;97:2007–11. [11] Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. New Engl J Med 1997;336:973–9. [12] Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease. Circulation 1998;97:425–8. [13] Scandinavian Simvastatin Survival Study Group. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344:1383–9. [14] Downs JR, Clearfield M, Weis S et al., for the AFCAPS / TexCAPS Research Group. Primary prevention of acute coronary events with lovastatin in men and women with average cholesterol levels. Results of AFCAPS / TexCAPS. J Am Med Assoc 1998;279:1615– 22. [15] The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. New Engl J Med 1998;339:1349– 57. [16] Szczeklik A, Musial J, Undas A et al. Inhibition of thrombin generation by simvastatin and lack of additive effects of aspirin in patients with marked hypercholesterolemia. J Am Coll Cardiol 1999;33:1286–93. [17] Rossen RD. HMG-CoA reductase inhibitors: a new class of antiinflammatory drugs? J Am Coll Cardiol 1997;30:1218–9. [18] Vaughan CJ, Murphy MB, Buckley BM. Statins do more than just lower cholesterol. Lancet 1996;348:1079–82. [19] Strandberg TE, Vanhanen H, Tikkanen MJ. Effect of statins on C-reactive protein in patients with coronary artery disease. Lancet 1999;353:118–9. [20] Rosenson RS, Tangney CC, Casey LC. Inhibition of proinflammatory cytokine production by pravastatin. Lancet 1999;353:983–4. [21] Ridker PM, Rifai N, Pfeffer MA, Sacks F. Braunwald E; for the Cholesterol and Recurrent Events (CARE) Investigators. Long-term effects of pravastatin on plasma concentration of C-reactive protein. Circulation 1999;100:230–5. [22] Williams JK, Sukhova GK, Herrington DM, Libby P. Pravastatin has cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys. J Am Coll Cardiol 1998;31:684–91. ´ [23] Bustos C, Hernandez-Presa A, Ortego M et al. HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis. J Am Coll Cardiol 1998;32:2057– 64. [24] Weber C, Erl W, Weber KSC, Weber PC. HMG-CoA reductase inhibitors decrease CD11b expression and CD11b-dependent adhesion of monocytes to endothelium and reduce increased adhesives of monocytes isolated from patients with hypercholesterolemia. J Am Coll Cardiol 1997;30:1212–7. [25] Bellosta S, Via D, Canavesi M et al. HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages. Arterioscler Thromb Vasc Biol 1998;18:1671–8.
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253 [26] Aylward M, Maddock J, Wheeldon R, Parker RJ. A study of the influence of various antirheumatic drug regimens on serum acutephase proteins, plasma tryptophan, and erythrocyte sedimentation rate in rheumatoid arthritis. Rheumatol Rehabil 1975;14:101–14. [27] Dixon JS, Bird HA, Sitton NG, Pickup ME, Wright V. C-reactive protein in the serial assessment of disease activity in rheumatoid arthritis. Scand J Rheumatol 1984;13:39–44. [28] Bruno JJ. Inflammation, aspirin, and the risk of cardiovascular disease. New Engl J Med 1997;337:422, (Letter).
253
[29] Ikonomidis I, Andreotti F, Enonomou E, Stefanadis C, Toutouzas P, Nihoyannopoulos P. Increased proinflammatory cytokines in patients with chronic stable angina and their reduction by aspirin. Circulation 1999;100:793–8. ´ ´ [30] de Miguel LS, de Frutos T, Gonzales-Fernandez F et al. Aspirin inhibits inducible nitric oxide synthase expression and tumor necrosis factor-a release by cultured smooth muscle cells. Eur J Clin Invest 1999;29:93–9.
Anti-inflammatory effects of simvastatin in subjects with hypercholesterolemia Jacek Musial*, Anetta Undas, Piotr Gajewski, Milosz Jankowski, Wojciech Sydor, Andrzej Szczeklik Department of Medicine, Jagiellonian University School of Medicine, Skawinska 8, 31 -066 Krakow, Poland Received 19 July 2000; received in revised form 24 October 2000; accepted 28 October 2000
Abstract Aims: Beneficial effects of statins in preventing cardiovascular events may depend, at least in part, on their anti-inflammatory action. The aim of the study was to assess the influence of simvastatin and aspirin on serum levels of C-reactive protein (CRP), tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) in hypercholesterolemic subjects. Methods and results: In 33 asymptomatic men with total cholesterol (TC) .6.5 mmol l 21 and in 25 men with coronary heart disease and borderline-high cholesterol levels (between 5.2 and 6.5 mmol l 21 ) chronically treated with low-dose aspirin (75 mg / d), serum levels of CRP, TNF-a, IL-6, and IL-8 were determined before and after a 3-month simvastatin therapy (20–40 mg daily). In the former group, these markers of inflammation were also measured before and after a 2-week treatment with aspirin (300 mg / d), implemented prior to and in combination with simvastatin. A distinct reduction of CRP and TNF-a was found in both groups; IL-6 levels were decreased only in subjects with marked hypercholesterolemia. Aspirin had no effect on the anti-inflammatory action of simvastatin. Conclusions: In men with hypercholesterolemia simvastatin treatment lowers serum levels of CRP and proinflammatory cytokines. Low-dose aspirin does not add to the anti-inflammatory action of simvastatin. 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Simvastatin; Aspirin; Hypercholesterolemia; C-reactive protein; Proinflammatory cytokines
1. Introduction Atherosclerosis is now considered an inflammatory disease [1]. This particular type of arterial inflammation may be initiated and sustained by several factors. Elevated levels of plasma cholesterol, particularly low-density lipoprotein (LDL) cholesterol, are one of the most important risk factors for cardiovascular disease [2]. In the arterial wall LDL particles may be modified and taken up by macrophage scavenger
*Corresponding author. Tel.: 148-12-656-2840; fax: 148-12-6565786. E-mail address: [email protected] (J. Musial).
receptors. This leads to the foam cell formation and initiation of an inflammatory response [1,3]. C-reactive protein (CRP) is a major acute phase protein and a known clinical marker of inflammation. Its production in the liver remains under strict control of proinflammatory interleukin-1-type (e.g. tumor necrosis factor-a-TNF-a) and interleukin-6-type (e.g. interleukin-6–IL-6) cytokines [4]. Plasma concentration of CRP correlates with severity and extent of the atherosclerotic process [5] and may mediate itself the inflammatory process in the arterial wall [6]. Higher CRP levels could serve as a prognostic marker of coronary events in survivors of myocardial infarction and in patients with stable or unstable angina [7–9]. Moreover, CRP level predicts the first
0167-5273 / 01 / $ – see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 00 )00439-3
248
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
myocardial infarction and other atherothrombotic events in apparently healthy men [10–12]. Lowering of serum cholesterol with statins (3hydroxy-3-methylglutaryl-CoA reductase inhibitors), used in the primary and secondary prevention of coronary heart disease (CHD), reduces the risk of acute cardiovascular events and prolongs survival. Beneficial effects of statins extend across a wide range of serum cholesterol levels [13–15]. Recently, besides lipid-lowering action, nonlipid effects of statins, including antithrombotic [16] and anti-inflammatory [17] properties, have been strongly suggested [5,18]. Two preliminary, recent reports indicate that treatment with statins could lower plasma CRP levels in hyperlipidemic coronary patients [19], and decrease TNF-a and IL-6 concentrations in lipopolysaccharide (LPS)-stimulated blood samples [20]. Therefore, we decided to study in detail the effects of simvastatin treatment on plasma levels of CRP and proinflammatory cytokines (TNF-a and IL-6) in subjects with elevated serum total cholesterol (TC) concentrations. We also compared the effects of simvastatin versus that of aspirin. For this purpose, in a group of patients with marked hypercholesterolemia 21 (TC .6.5 mmol l ), we investigated the action of a 2-week treatment with aspirin before and after lowering cholesterol levels by simvastatin.
2. Methods The design of the study (Group 1) has been described in detail elsewhere [16]. In brief, the study enrolled 33 healthy male volunteers with a mean age of 47.4 years (range, 34 to 61 years) whose serum TC was .6.5 mmol l 21 , LDL cholesterol .3.6 mmol l 21 , and fasting triglycerides ,4.6 mmol l 21 during 6 weeks of treatment with the National Cholesterol Education Program Step 1 diet. Subjects were eligible if they have fasting plasma glucose level ,6.7 mmol l 21 , creatinine ,120 mmol l 21 , absence of symptomatic CHD or congestive heart failure or other severe chronic illness (including a history of venous thromboembolism). Subjects were not allowed to take aspirin or any agents interfering with hemostasis for at least 4 weeks before entering the study. Twenty
men smoked regularly (on average, 10 cigarettes daily). During the first 2 weeks of the study, subjects received 300 mg of aspirin daily (Phase I). Then, they were given simvastatin for 12 weeks (Phase II). The starting dose of the drug was 20 mg taken once at bedtime. The dose was doubled after 4 weeks, if the reduction in TC was less than 1 mmol l 21 . The maximal dose of simvastatin was 40 mg a day. Finally, aspirin 300 mg / d was added to simvastatin for the additional 2 weeks (Phase III). Haemostatic parameters and lipid profiles, analysed in this group, have already been published [16]. Group 2 consisted of 25 age-matched patients (range; 39 to 64 years) with borderline-high cholesterol levels (between 5.2 and 6.5 mmol l 21 ; median 5.91 mmol l 21 ). For inclusion, patients were required to have a history (at least 6 month before entering the study) of myocardial infarction or hospitalization due to unstable angina pectoris. Other inclusion and exclusion criteria were identical to those accepted for the subjects with marked hypercholesterolemia. Six men were current smokers (on average, 10 cigarettes daily). Patients were regularly treated with 75 mg of aspirin daily. Simvastatin, as a study agent, was given for 12 weeks at a dose of 20 mg a day. The therapeutic goal was to reduce TC below 5.2 mmol l 21 . At the study entry, none of the men enrolled had any clinical signs or symptoms of infection. Informed consent to participate in the study was obtained from all individuals. Fasting venous blood samples were collected with minimal stasis between 8 a.m. and 10 a.m. Serum samples for inflammatory markers were frozen at 2808C until assayed, but no longer than 12 months. Serum levels of CRP were measured nephelometrically (Behring, Marburg, Germany) using a sensitive assay (detection limit 0.16 mg l 21 of the protein). Reported intra-assay and inter-assay coefficients of variation were 4% and 2.6%, respectively. Concentrations of TNFa and IL-6 in serum were determined by chemiluminescent enzyme immunometric assays, using the IMMUNOLITE Automated Analyzer (DPC, Los Angeles, USA). Intra-assay and interassay coefficients of variation were 3.5% and 6.5%, and 7.9% and 3.6%, respectively. The detection limits of the assays were approximately 1.7 pg ml 21 for TNF-a and 1 pg ml 21 for IL-6). In subjects with
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
marked hypercholesterolemia all the markers of inflammation were determined 4 times. They were performed at baseline (week 0), after 2 weeks of aspirin administration (Phase I), after a 3-month simvastatin treatment (Phase II) and finally, after the 2 weeks of combined aspirin and simvastatin treatment (Phase III). In the Group 2 determinations were performed twice: at baseline and at the end of 12 week simvastatin treatment. All samples were coded in order to blind the laboratory personnel regarding subject’s name and phase of treatment.
3. Statistical analysis Since inflammatory variables in Group 1 were not normally distributed, repeated measure analysis was performed by Friedman’s test, followed by paired analysis using Wilcoxon matched pairs test. Correlations were assessed by Spearman rank order test. In Group 2 inflammatory variables also did not follow normal distribution and were compared by Wilcoxon matched pairs test. Values were expressed as median and interquartile range. Statistical significance was accepted at a level of P,0.05. Calculations were performed on a PC computer using Statistica (Statsoft Inc., Tulsa, OK, USA) software.
4. Results As previously reported [16], in men with marked hypercholesterolemia (Group 1), a 3-month simvastatin treatment led to a reduction of TC by 31% (from a median of 7.85–5.43 mmol l 21 ; P , 0.00001), LDL cholesterol by 41% (from a median of 5.57–3.26 mmol l 21 ; P , 0.00001), and triglycerides by 26%
249
(from a median of 2.32–1.72 mmol l 21 ; P , 0.00001), whereas HDL cholesterol was raised by 7.5% (from a median of 1.34–1.44 mmol l 21 ; P . 0.05). Aspirin did not influence serum CRP concentrations. Treatment with simvastatin for 3 months was associated with a significant reduction in CRP levels by about 50%. A similar fall (by about 60%) of CRP levels was found after the combined treatment with aspirin (300 mg / d) added for 2 weeks to simvastatin. However, the difference between Phase II and Phase III values did not reach the level of statistical significance (Table 1, Fig. 1A). In none of the subjects studied the level of CRP exceeded 10 mg l 21 . There was a significant correlation between CRP level at baseline and its relative change after simvastatin treatment (R50.70, P , 0.0001). It suggests a greater reduction in serum CRP levels by simvastatin in subjects with higher baseline values of this marker. Men with CRP.1 mg l 21 (n518) had a 12-fold higher chance of reduction in CRP levels after a 3-month simvastatin therapy (odds ratio [OR]5 12.00; 95% confidence interval [CI]: 2.23–64.65; P 5 0.003). After the combined treatment with aspirin and simvastatin this effect was even more pronounced (OR514.00; 95% CI: 1.86–104.85; P 5 0.008). Interestingly, serum TNF-a levels were decreased slightly, but significantly, after aspirin alone. Simvastatin treatment reduced TNFa levels by about 20% in comparison with the initial values. Addition of aspirin to simvastatin for the additional 2 weeks reduced TNFa values as compared to baseline and the levels measured after aspirin alone. This combination, however, did not significantly change TNFa levels, decreased previously by simvastatin treatment (Table 1, Fig. 1A). Aspirin had no effect on serum IL-6 levels, while
Table 1 Influence of aspirin and simvastatin on markers of inflammation in patients with marked hipercholesterolemia (Group 1)a Variables
At baseline
After aspirin (Phase I)
After simvastatin (Phase II)
After simvastatin1aspirin (Phase III)
P
C-reactive protein (mg l 21 ) TNF-a (pg?ml 21 ) Interleukin-6 (pg?ml 21 )
1.02 (0.44; 2.06) 4.9 (4.2; 7.9) 4.6 (2.7; 10.2)
1.04 (0.49; 1.51) 4.6 (4.0; 6.5)† 4.1 (3.0; 12.2)
0.52 (0.37; 1.35)* 4.0 (3.6; 4.3)† 2.7 (2.1; 3.4)*
0.39 (0.27; 0.89)†‡ 3.9 (3.6; 4.1)†§ 2.2 (2.0; 4.05)†‡
0.002 ,0.00001 0.0004
a
Values are median (quartile 1;3). Friedman’s test was applied followed by paired analysis using Wilcoxon test: *, P,0.05 as compared to baseline; †, P , 0.01 as compared to baseline; ‡, P , 0.01 as compared to phase I; §, P , 0.05 as compared to phase I.
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
250
Fig. 1. Concentrations (medians) of C-reactive protein (CRP), tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6) during simvastatin or / and aspirin treatment, expressed as a percentage of baseline values (100%). Bars denote interquartile ranges. a – changes in men with marked hypercholesterolemia – group 1 (n533) b – changes in men with borderline-high hypercholesterolemia – group 2 (n525).
simvastatin reduced it by about 40% as compared to the pretreatment values. After two additional weeks of combined aspirin and simvastatin administration IL-6 levels fell by more than 50%. There was no difference between Phase II and Phase III values (Table 1, Fig. 1A). Three-month simvastatin treatment of CHD patients with borderline high hypercholesterolemia (Group 2) led to the reduction of TC by about 25% (from a median of 5.91–4.39 mmol l 21 ; P , 0.0001), LDL-cholesterol by about 40% (from a median of 3.71–2.23 mmol l 21 ; P , 0.0001), while the levels of HDL-cholesterol and triglycerides remained unchanged. At the same time CRP levels decreased by more than 25% (Table 2) These changes were
accompanied by a slight but significant fall in the TNF-a levels, while serum IL-6 levels showed no change. There was no significant difference between Group 1 and Group 2 baseline levels of inflammatory markers studied, even if Group 2 was on chronic low-dose aspirin treatment. Interestingly, however, simvastatin had an almost four-fold greater chance to reduce TNF-a levels in subjects with marked hypercholesterolemia than in these with borderline-high TC values (OR53.6; 95% CI: 1.08–11.93). There were no similar relationships with regard to the remaining markers of inflammation. No correlation could be found between CRP, TNFa, and IL-6 values, before as well as after drug
Table 2 Influence of simvastatin on markers of inflammation in patients with moderate hipercholesterolemia (Group 2) under long-term aspirin treatment (75 mg / d)a Variables 21
C-reactive protein (mg l ) TNF-a (pg?ml 21 ) Interleukin-6 (pg?ml 21 ) a
At baseline
After simvastatin
P
1.61 (0.80; 2.88) 4.3 (4.0; 8.4) 2.9 (2.0; 16.3)
1.17 (0.31; 2.02) 4.0 (4.0; 4.5) 3.9 (2.0; 25.3)
0.011 0.015 N.S.
Values are median (quartile 1;3). Wilcoxon test was applied for statistical analysis.
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
interventions in either group. There was also no relation between lipid and inflammatory parameters, with an exception of a significant correlation between baseline TNF-a and TC levels in Group 1 (R 5 0.67; P , 0.0001). There was no difference in markers of inflammation depending on the smoking status in both groups. Change in serum levels of CRP, TNF-a, and IL-6 was not related to the dose of simvastatin given to the patients in Group 1; nine received 40 mg of the drug daily.
5. Discussion We have shown that in men with hypercholesterolemia a 3-month simvastatin treatment significantly reduces serum markers of inflammation. To our knowledge, no previous study has sought to explore changes in all the 3 markers: CRP, TNF-a and IL-6, measured in peripheral venous blood in subjects treated with statins. The significant reduction was particularly evident in subjects with markedly elevated cholesterol levels (.6.5 mmol l 21 ), in whom CRP, TNF-a, and IL-6, all decreased substantially. Less pronounced, but significant reductions in CRP and TNF-a were also observed in CHD patients with borderline high cholesterol-levels, who were already treated with low-dose aspirin. These results corroborate preliminary data, published by Strandberg et al. [19] and Rosenson et al. [20]. However, the former analysis was performed after 8 weeks of treatment with either atorvastatin or simvastatin given in the blinded fashion in a study, which is still underway. In the latter, reductions of TNF-a and IL-6 were observed in 7 subjects taking pravastatin, another HMG-CoA inhibitor, for 7 weeks but only when their blood was stimulated in vitro by lipopolysaccharide. No change in the IL-1b, IL-8 and IL-1 receptor antagonist was noted [20]. Recently, Ridker et al. [21] reported a reduction in plasma concentrations of CRP in participants of the CARE trial after 5 years of treatment with pravastatin. Beneficial effects of statins in atherosclerosis extend probably beyond their cholesterol-lowering action. Anti-inflammatory properties may be particularly important in protecting vessel wall and preventing acute vascular episodes. Evidence from ex-
251
perimental and human studies indicate that statins indeed possess such properties, which are likely to be independent from lipid-lowering effects [7,22]. Indeed, in a recent study Ridker et al. [21] could not show any correlation between lipid-lowering action of pravastatin and its ability to lower CRP levels. In experimental animals, pravastatin and atorvastatin reduce arterial inflammation [22,23]. On the other hand, lovastatin and simvastatin decrease monocyte CD11b surface expression and monocyte adhesion to endothelium in hypercholesterolemic patients [24]. In addition, fluvastatin could inhibit the secretion of matrix metalloproteinases from macrophages, and by this effect possibly prevent atherosclerotic plaque disruption [25]. The mechanism of anti-inflammatory action of statins has not been elucidated. Some evidence points to the decreased production of mevalonate and isoprenoids, the early products generated by HMG-CoA reductase [17,25]. The effect of simvastatin on CRP levels in marked hypercholesterolemia appears to depend on the initial levels of this protein. This significant correlation, found between the baseline concentrations of CRP and its statin-induced reduction, may be of clinical importance, particularly, in the light of the finding that patients with the highest serum levels of CRP prior to treatment with pravastatin had the highest risk for recurrent coronary events [7]. Further studies are needed to show whether clinical benefits from statin treatment are related to initial levels of markers of inflammation and degree of their reduction. Two-week treatment with 300 mg of aspirin per day did not influence the concentrations of serum inflammation markers, except for a slight fall in the TNF-a levels. No enhancement of anti-inflammatory effect was observed when aspirin was added for 2 weeks to simvastatin. This study was not designed to assess the effects of chronic aspirin treatment on serum markers of inflammation. For this reason we are unable to explain whether a less pronounced anti-inflammatory effect of simvastatin in patients with borderline-high cholesterol depends on an additional effect of low-dose aspirin treatment, or is a result of lower total cholesterol levels. While elder studies suggested that aspirin had no influence on CRP levels [26–28], a recent report showed 6 weeks of aspirin treatment in a daily
252
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253
dose of 300 mg lowers plasma levels of CRP, IL-6 and macrophage colony stimulating factor in patients with coronary heart disease [29]. In cell culture aspirin is able to inhibit TNF-a release [30]. Interestingly, aspirin has been shown to be particularly effective in preventing the first myocardial infarction in men with the highest C-reactive protein values [11]. The limitation of the current study is the absence of placebo group. However, most of subjects from group 1 have at least 2 coronary risk factors, in addition to marked hypercholesterolemia, while those from group 2 have documented coronary artery disease, we considered it unethical to postpone cholesterol-lowering treatment.
6. Conclusions In subjects with a broad range of cholesterol levels simvastatin exerts a marked anti-inflammatory effect. This could explain, at least in part, be responsible for the clinical effectiveness of the drug in lowering the risk of acute cardiovascular complications and prolonging life. It has been suggested that anti-inflammatory agents may provide clinical benefits in preventing cardiovascular disease [11]. Simvastatin certainly belongs to such agents.
References [1] Ross R. Atherosclerosis – an inflammatory disease. New Engl J Med 1999;340:115–26. [2] National Cholesterol Education Program. Second report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). Circulation 1994;89:1329–445. [3] Steinberg D. Low-density lipoprotein oxidation and its pathobiological significance. J Biol Chem 1997;272:20963–6. [4] Baumann H, Gauldie J. The acute phase response. Immunol Today 1994;15:74–80. ¨ ¨ [5] Heinrich J, Schulte H, Schonfeld R, Kohler E, Assmann G. Association of variables of coagulation, fibrinolysis and acute-phase with atherosclerosis in coronary and peripheral arteries and those arteries supplying the brain. Thromb Haemost 1995;73:374–9. [6] Lagrand WK, Visser CA, Hermens WT et al. C-reactive protein as a cardiovascular risk factor. More then an epiphenomenon? Circulation 1999;100:96–102. [7] Ridker PM, Rifai N, Pfeffer MA et al., for the Cholesterol and Recurrent Events (CARE) Investigators. Inflammation, pravastatin and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Circulation 1998;98:839– 44.
[8] Haverkate F, Thompson SG, Pyke SD, Gallimore JR, Pepys MB, for the European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. Production of C-reactive protein and risk of coronary events in stable and unstable angina. Lancet 1997;349:462–6. [9] Verheggen PWHM, de Maat MPM, Manger Cats V et al. Inflammatory status as a main determinant of outcome in patients with unstable angina, independent of coagulation activation and endothelial cell function. Eur Heart J 1999;20:567–74. [10] Ridker PM, Glynn RJ, Hennekens CH. C-reactive protein adds to the predictive value of total and HDL cholesterol in determining risk of first myocardial infarction. Circulation 1998;97:2007–11. [11] Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. New Engl J Med 1997;336:973–9. [12] Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease. Circulation 1998;97:425–8. [13] Scandinavian Simvastatin Survival Study Group. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344:1383–9. [14] Downs JR, Clearfield M, Weis S et al., for the AFCAPS / TexCAPS Research Group. Primary prevention of acute coronary events with lovastatin in men and women with average cholesterol levels. Results of AFCAPS / TexCAPS. J Am Med Assoc 1998;279:1615– 22. [15] The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. New Engl J Med 1998;339:1349– 57. [16] Szczeklik A, Musial J, Undas A et al. Inhibition of thrombin generation by simvastatin and lack of additive effects of aspirin in patients with marked hypercholesterolemia. J Am Coll Cardiol 1999;33:1286–93. [17] Rossen RD. HMG-CoA reductase inhibitors: a new class of antiinflammatory drugs? J Am Coll Cardiol 1997;30:1218–9. [18] Vaughan CJ, Murphy MB, Buckley BM. Statins do more than just lower cholesterol. Lancet 1996;348:1079–82. [19] Strandberg TE, Vanhanen H, Tikkanen MJ. Effect of statins on C-reactive protein in patients with coronary artery disease. Lancet 1999;353:118–9. [20] Rosenson RS, Tangney CC, Casey LC. Inhibition of proinflammatory cytokine production by pravastatin. Lancet 1999;353:983–4. [21] Ridker PM, Rifai N, Pfeffer MA, Sacks F. Braunwald E; for the Cholesterol and Recurrent Events (CARE) Investigators. Long-term effects of pravastatin on plasma concentration of C-reactive protein. Circulation 1999;100:230–5. [22] Williams JK, Sukhova GK, Herrington DM, Libby P. Pravastatin has cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys. J Am Coll Cardiol 1998;31:684–91. ´ [23] Bustos C, Hernandez-Presa A, Ortego M et al. HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis. J Am Coll Cardiol 1998;32:2057– 64. [24] Weber C, Erl W, Weber KSC, Weber PC. HMG-CoA reductase inhibitors decrease CD11b expression and CD11b-dependent adhesion of monocytes to endothelium and reduce increased adhesives of monocytes isolated from patients with hypercholesterolemia. J Am Coll Cardiol 1997;30:1212–7. [25] Bellosta S, Via D, Canavesi M et al. HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages. Arterioscler Thromb Vasc Biol 1998;18:1671–8.
J. Musial et al. / International Journal of Cardiology 77 (2001) 247 – 253 [26] Aylward M, Maddock J, Wheeldon R, Parker RJ. A study of the influence of various antirheumatic drug regimens on serum acutephase proteins, plasma tryptophan, and erythrocyte sedimentation rate in rheumatoid arthritis. Rheumatol Rehabil 1975;14:101–14. [27] Dixon JS, Bird HA, Sitton NG, Pickup ME, Wright V. C-reactive protein in the serial assessment of disease activity in rheumatoid arthritis. Scand J Rheumatol 1984;13:39–44. [28] Bruno JJ. Inflammation, aspirin, and the risk of cardiovascular disease. New Engl J Med 1997;337:422, (Letter).
253
[29] Ikonomidis I, Andreotti F, Enonomou E, Stefanadis C, Toutouzas P, Nihoyannopoulos P. Increased proinflammatory cytokines in patients with chronic stable angina and their reduction by aspirin. Circulation 1999;100:793–8. ´ ´ [30] de Miguel LS, de Frutos T, Gonzales-Fernandez F et al. Aspirin inhibits inducible nitric oxide synthase expression and tumor necrosis factor-a release by cultured smooth muscle cells. Eur J Clin Invest 1999;29:93–9.