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the Departments of Vascular Surgery (B.A.N.V., F.L.M.) and Experimental Cardiology Laboratory (J.A.F.K., D.P.V.d.K., E.V., E.B., G.P.), University Medical Centre Utrecht, Utrecht, The Netherlands
the Cardiovascular Pathology (A.C.v.d.W.), Academic Medical Centre, University of Amsterdam, The Netherlands
the Interuniversity Cardiology Institute of The Netherlands, Utrecht, The Netherlands (D.P.V.d.K., A.S.)
the Department of Vascular Surgery (J.P.P.M.d.V.), St. Antonius Hospital Nieuwegein, The Netherlands
the Biomedical Research (J.H.V.)
de Nederlandse Organisatie voor toegepast-natuurwetenschappelijk onderzoek Quality of Life, Leiden, The Netherlands
the Department of Cardiovascular Pathology (R.V.), Armed Forces Institute of Pathology, Washington, DC, USA.
Abstract
Background and Purpose— Anti-inflammatory qualities are held partially responsible for the reduction of cardiovascular events after statin treatment. We examined the phenotype of carotid atherosclerotic plaques harvested during carotid endarterectomy in relation to the previous use of different statins prescribed in clinical practice.
Methods— Three hundred and seventy-eight patients were included. Atherosclerotic plaques were harvested, immunohistochemically stained and semiquantitively examined for the presence of macrophages (CD68), smooth muscle cells, collagen and fat. Adjacent atherosclerotic plaques were used to study protease activity and interleukin levels. Patients’ demographics were recorded and blood samples were stored.
Results— Serum cholesterol, low-density lipoprotein, apolipoprotein B, and C-reactive protein levels were lower in patients treated with statins compared with patients without statin treatment. Atheromatous plaques were less prevalent in patients receiving statins compared with patients without statin therapy (29% versus 42%, P=0.04). An increase of CD68 positive cells was observed in patients receiving statins compared with nonstatin treatment (P=0.05). This effect was specifically related to atorvastatin treatment. In patients treated with atorvastatin, the increased amount of CD68 positive cells were not associated with increased protease activity. In contrast, a dose-dependent decrease in protease activity was shown in the atorvastatin group. Interleukin 6 expression was lower in plaques obtained from patients treated with statins (P=0.04).
Conclusions— Statin use may exert pleiotropic effects on plaque phenotype. However, not the presence of macrophages but activation with subsequent protease and cytokine release may be attenuated by statin use.
Key Words: carotid endarterectomy inflammation pharmacology
Introduction
Hyperlipidemia is a risk factor for cardiovascular disease and is related to the increase of plaque atheroma.1 Treatment with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) clearly results in reduced cardiovascular-related mortality and morbidity.2,3
Next to the lipid-lowering effects, statin treatment has potential pleiotropic effects by modulating various inflammatory responses that are involved in the initiation and progression of atherosclerotic disease.4 Statin-induced reductions of serum interleukin (IL) levels, C-reactive protein (CRP) and matrix metalloproteinase (MMP) activity have been described previously.3,5–9 However, pleiotropic effects of statin treatment on atherosclerotic plaque characteristics have mainly been described in animal studies. Pravastatin has been investigated in many experimental cardiovascular studies.10–13 The modifying effects of atorvastatin or simvastatin on human plaque characteristics are relatively unexplored. A prospective longitudinal study including a nontreated placebo group would be the best design to answer our research question. However, considering the beneficial effects of statins in a population experiencing atherosclerotic cardiovascular disease, the compliance of the practitioner and patient would be a major limitation.
In this retrospective study, we analyzed the pleiotropic effects of 3 regularly prescribed statins on plaque characteristics in 378 patients undergoing carotid endarterectomy (CEA).
Methods
Baseline Data
Patients have been included in the longitudinal cohort bio-bank study Athero-Express. The study design of Athero-Express has been published previously.14
Briefly, all patients receiving operative treatment for carotid artery disease in the participating centers are enrolled. Patients may have been symptomatic or asymptomatic. Operation is indicated based on the recommendations as published by ACST and NASCET.15,16 Patients with a terminal malignancy and those who are referred back to a hospital outside The Netherlands immediately after surgery are excluded. The Medical Ethical Committees of the participating hospitals have approved of the study.
patients included in Athero-Express extensive baseline patient characteristics are available (history of cardiovascular disease, medication, etc).14 In cases of doubt about medication use or dosage, the patient’s general practitioner or pharmacy was consulted. Statin dosages were recorded and divided into 2 categories: low-dose (simvastatin, pravastatin and atorvastatin, 10 and 20 mg) and high-dose (simvastatin and atorvastatin, 40 and 80 mg; pravastastin, 40 mg). All analyses have been performed in the same cohort of 378 patients. The total number of 378 plaques were all used for immunohistochemistry and protein isolation was available for 293 patients. IL levels were determined in all protein isolates. MMP activity levels were assessed in 133 randomly selected protein isolates.
Of the 378 patients, blood samples were available of 228 patients. Total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein B and high-sensitive CRP were assessed before surgery. Baseline patient characteristics are presented in Table 1.
Tissue Processing
The atherosclerotic plaque obtained during CEA was immediately processed and dissected in parts of 0.5 cm. The culprit lesion was fixated in formaldehyde 4%, paraffin embedded and used for histological stainings. The segments adjacent to the culprit lesion were immediately frozen in liquid nitrogen and stored at –80°C.
All stained sections were independently scored by 2 observers as described previously.17 Plaques stainings were categorized as follows:
Picro Sirius red; collagen staining using polarized light microscopy: (1) no or minor staining= staining along part of the luminal border; (2) moderate or heavy staining=staining along the entire luminal border.
CD68 positive cells: (1) absent or minor staining=negative or few scattered positive cells; (2) moderate or heavy staining=stained clusters of cells with >10 cells present.
-actin positive cells: (1) no or minor staining=discontinuous over the entire circumference with absent staining at parts of the circumference of the arterial wall; (2) positive cells along the entire circumference of the luminal border, with at least minor staining locally with few scattered cells.
Hematoxylin and elastin stains were used to assess overall morphology. The percentage of atheroma of the total area of the plaque was visually estimated using the picro Sirius red with polarized light and hematoxylin stains. Three groups were defined based on the percentage of atheroma in the plaque: fibrous plaques <10% fat, fibro-atheromatous 10% to 40% or atheromatous >40% fat.
Human leukocyte antigens (HLA-DR) staining was performed in patients with moderate or heavy CD68 stained plaques and treated with atorvastatin (n=20) or no statin (n=22). Three groups were defined based on the percentage of cells being activated in the plaque <25%, 25% to 75% or >75%.
The CD68 (macrophage) staining was also analyzed quantitatively by computerized analyses using Analysis software. The stained sections were scored quantitatively as a percentage of the plaque area.
The adjacent segments were used for protein isolation. Protein was isolated by 2 protocols. One part of segment 1 was treated with 1 mL Tripure Isolation Reagent (Boehringer Mannheim) according to the manufacturer’s protocol. The other part was dissolved in 1 mL 40 mmol TrisHCl (pH=7.5; 4°C) and centrifuged at max rpm after which the vials were stored at –80°C. Protein concentrations for both isolation techniques were measured according to the manufacture’s protocol (Bio-Rad Laboratories).
For a randomly selected subgroup of 133 plaques, MMP-2, MMP-8 and MMP-9 activities were measured in isolated protein using the Biotrak activity assays RPN 2631, RPN 2635 and RPN 2634, respectively (Amersham Biosciences).18,19 IL-6 and -8 were measured in 293 protein isolates using a multiplex suspension array system according to the manufacture’s protocol (Bio-Rad Laboratories).
Statistical Analyses
Statins were associated with plaque characteristics using a 2 and Fisher exact test. Additionally, a binary logistic regression analysis was used in cases of confounding. In the cases of nonparametric distributed continuous variables, comparison of the variables was performed by a Mann–Whitney test and a Kruskal Wallis test. For normal distributed parameters, t tests and Anova tests were used. Probability values of <0.05 were considered statistically significant.
Results
Patients who received statin therapy showed a higher prevalence of cardiovascular morbidity and risk factors in the past (Table 1). These risk factors were equally distributed among the 3 major statins described. We examined the relations between all baseline characteristics and the investigated markers for plaque phenotype to rule out confounding of baseline characteristics in the studied correlations between statin use and plaque phenotype. Overall, plaque phenotype (eg, amount of fat, protease activity and IL levels) was neither associated with any of the risk factors nor with clinical history (all P>0.10). CD68 positive stained cells were more prevalent in plaques obtained from patients with prior coronary intervention in the past and a tendency was noticed for hypertension (P=0.04 and P=0.06).
Baseline characteristics of serological assessments are presented in Table 2. Serum levels of cholesterol, LDL and apolipoprotein B as well as CRP were significantly lower among statin users (Table 2).
Atheromatous ("lipid rich") plaques were less prevalent in patients receiving statins compared with patients without statin therapy (29% versus 42%; P=0.04; Table 3). The prevalence of atheromatous plaques was not associated with statin type or dosage. A tendency toward a lower prevalence of atheromatous plaques was noticed for patients receiving statins for >1 year (no statin: 41.9% atheromatous plaques, <1 year statin 32.9% and >1 year statin 27.7%; P=0.07).
In contrast to the decreased level of unstable "lipid rich plaques," a significant increase in CD68 positive cells was observed in the statin group (P=0.05; Table 3 and Figure 1). To rule out confounding between statin use and the previously mentioned baseline characteristics that were related to CD68 positive cells (P<0.1; hypertension and prior coronary intervention), a binary logistic regression analysis was performed to identify the independent relations. Statin use was independently related to an increased number of CD68 positive cells in the plaque (P=0.02), whereas prior coronary intervention and high blood pressure were not (P=0.25 and 0.06).
These observations would appear to conflict with previous studies demonstrating that pravastatin treatment is related to lower macrophage content in the atherosclerotic plaque. Therefore, we decided to perform subgroup analyses for the different, frequently prescribed statins. Plaques derived from patients receiving atorvastatin showed significantly more CD68 positive cells than those receiving pravastatin (Table 3; P=0.01). Prolonged statin therapy (>1 year) and high dosage were not related to a decreased prevalence of CD68 positive cells (Figure 1).
The increased amount of CD68 positive cells in patients who received statin treatment was not associated with an increased MMP activity (Table 3). In contrast, patients treated with high doses of statin showed less protease activity compared with patients treated with low doses of statin (Figure 2A). In the nonstatin-treated group MMP-8 and -9 activities were associated with the degree of CD68 positive staining. The relation between CD68 staining and MMP activities was abolished in the atorvastatin-treated group but remained significant in the pravastatin-treated group (Figure 2B). Patients with strong CD68 staining and high dose atorvastatin revealed lower activity levels of MMP-8, and a tendency was shown for MMP-9 compared with patients with low dose and strong CD68 staining (P=0.02 and P=0.06; Figure 3). Statin use did not influence MMP-2 activity (data not shown).
IL-6 levels were lower in plaques obtained from patients who were on statin treatment compared with plaques derived from patients who did not receive statin treatment (P=0.04; Table 3). No significant differences were observed for IL-8 among patients groups (Table 3).
As an indication of activated CD68 cells we measured plaque HLA-DR expression in patients with high staining for CD68. No difference between the percentage of activated cells was observed between patients treated with atorvastatin and patients without statin treatment (P=0.99).
Besides the observed relations for statin treatment, other often prescribed medications in cardiovascular disease (angiotensin-converting enzyme inhibitors, calcium-antagonists, blockade) were not associated with plaque phenotype (data not shown).
Discussion
Statin treatment of cardiovascular patients results in improved clinical outcome and reduction of serum LDL and CRP levels.3,20,21 Recently, a significant difference in hazard ratio for cardiovascular events was reported for symptomatic patients pretreated with statins at the time of carotid endarterectomy.22 Reduction of serum IL levels has also been described in relation to statin treatment.8,23 However, statin-related effects on plaque characteristics have only been described in a limited number of studies that were limited by the number of specimens analyzed.10–13,24 In this retrospective study, we analyzed the inflammatory status of atherosclerotic plaques obtained during CEA in relation to statin therapy.
It has been acknowledged that inflammatory mechanisms couple dyslipidemia to atheroma formation.25 Statin treatment results in reduced serum cholesterol levels, impairs inflammatory processes and might stabilize atheromatous plaques.12,24,26 The current study confirms the earlier experimental observations that statin treatment results in lower fat content of atherosclerotic plaques. However, in contrast with previous studies, we found a slight but significant increase of CD68 positive cells in plaques obtained from statin-treated patients which was mainly attributable to the inclusion of the atorvastatin-treated patients. This was even more surprising considering the recently published clinical trial demonstrating the beneficial outcome for atorvastatin users.27 Our observation does not imply that the inflammatory response is enhanced in atherosclerotic tissue of atorvastatin-treated patients. CD68 is considered a pan macrophage marker and does not provide information about the functional status of macrophages. The question rises whether the CD68 positive cells in patients treated with statins are inactivated. Within the atherosclerotic plaque, statins could suppress the production of cytokines and MMP by CD68 positive cells. This hypothesis is supported by our observations and is also substantiated by earlier fundamental research.12,24 We cannot explain the increased number of CD68 positive cells in the atorvastatin group. Chemokine expression is attenuated after atorvastatin treatment and increased adhesion of monocytes is not a likely explanation.28 In an experimental model it has been shown that statin treatment reduced apoptosis in macrophages which could theoretically increase relative macrophage content.29
Heavy CD68 stained plaques correlated with increased protease activity. This association was eliminated in atorvastatin- but not in pravastatin-treated patients. Moreover, IL-6 levels were lowered among patients treated with statins. It could be suggested that the pleiotropic effect of statin treatment is more reflected on a macrophage activation than on a macrophage recruitment level. For this reason, we studied HLA-DR expression, as an indication for macrophage activation. However, any conclusive remarks regarding inactive macrophages merit careful consideration when based on immunohistochemistry. HLA-DR expression did not differ significantly between statin users and nonstatin users. This observation makes a dramatic effect of statins on HLA-DR unlikely. However, HLA-DR expression on inflammatory cells on immune activation is not a de novo ("on-off") feature; on activated cells the number of HLA-DR molecules increases drastically. Such a phenomenon may result in increased staining intensity, which we were unable to demonstrate, but which does not allow the tracing of discrete differences in expression (and hence activation).
Besides the above-mentioned possibilities to explain the observed differences in CD68 staining, hypercholesterolemia itself could be an explanation. Significantly more patients in the statin group had a prior history of hypercholesterolemia. However, baseline serum lipid spectra are lower in the statin group compared with the nonstatin group. We also noted significantly lower serum CRP (activated plaque macrophages could produce CRP) levels in patients treated with statins compared with patients with no statin. Strictly hypothetically, the observed increased number of macrophages in the atherosclerotic plaques could be necessary for organizing plaque lipid remains.
What do these observations mean for clinical practice First, imaging modalities are being developed to visualize the vulnerable plaque and the patient who is at risk for plaque rupture and subsequent cardiovascular symptoms. The present study suggests that in patients receiving statins, the inflammatory status of the atherosclerotic plaque should not be based on the number of inflammatory cells which may not always relate to plaque vulnerability.
Secondly, long-term statin treatment may be necessary to optimally influence plaque phenotype. We report a tendency that the statin associated effects on plaque characteristics are more pronounced in patients receiving statins longer than 1 year (P=0.07). The reported effect of statins on LDL serum levels and incidence of myocardial infarction has also been observed after prolonged treatment.30 Sever et al also reports significant differences in hazard ratio between atorvastatin and placebo occurring at 2-year follow-up.31
Limitations
This study is based on retrospective data. No causality can be inferred from the current data.
Besides the modifying effect of therapy duration, we also observed a dose-response effect. We report a dose-response relation between plaque MMP levels and statin dose. This relation was not evident for IL-6 and IL-8. The dosages among the different statins were not equally distributed and could be of influence because the reported effects on inflammation differ among statins.
Our selected population could induce a bias. Higher rates of prior coronary intervention, hypertension, diabetes and previous cardiovascular events were reported in the statin-treated group. Patients with these characteristics would be expected to have more advanced and more severe atherosclerosis and quite likely, greater inflammation and other biological derangements so typical of atherosclerosis. However, we excluded confounding and this cannot explain the differences observed among simvastatin, pravastatin and atorvastatin.
Sampling error could be an issue. Adjoining tissue showed less protease and IL-6 activity than would be expected from the amount of macrophages in the section examined by histology. Plaque characteristics may be heterogeneous within a short distance. Therefore, expression of protein or RNA may not always reflect the association with an immunohistochemically assessed phenotype. This limitation merits consideration when a limited number of samples is used. However, the relation we observed in control tissues in this and previous studies between MMP-9 and CD68 stainings, for instance, supports the idea that inflammatory markers are present in both adjacent segments.
Any conclusions regarding inactive macrophages cannot be drawn from the immunochemistry study alone. However, the finding that an increase in macrophage content was not associated with increased activity of MMP-8 and MMP-9 in the atorvastatin group supports the immunohistochemical observations.
Summary
Statin therapy was associated with a decreased prevalence of carotid atheromatous plaques. The number of CD68 positive cells increased slightly in patients receiving atorvastatins. Not the presence of macrophages but activation with subsequent protease and cytokine release seem attenuated by statin use.
Acknowledgments
Disclosures
None.
References
Ross R, Harker L. Hyperlipidemia and atherosclerosis. Science. 1976; 193: 1094–1100.
Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994; 344: 1383–1389.
Nissen SE, Tuzcu EM, Schoenhagen P, Crowe T, Sasiela WJ, Tsai J, Orazem J, Magorien RD, O’Shaughnessy C, Ganz P. Statin therapy, LDL cholesterol, C-reactive protein, and coronary artery disease. N Engl J Med. 2005; 352: 29–38.
Halcox JP, Deanfield JE. Beyond the laboratory: clinical implications for statin pleiotropy. Circulation. 2004; 109: II42–II48.
Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685–1695.
Libby P. Lipid-lowering therapy stabilizes plaque, reduces events by limiting inflammation. Am J Manag Care. 2002; Suppl:1, 4.
Nawawi H, Osman NS, Yusoff K, Khalid BA. Reduction in serum levels of adhesion molecules, interleukin-6 and C-reactive protein following short-term low-dose atorvastatin treatment in patients with non-familial hypercholesterolemia. Horm Metab Res. 2003; 35: 479–485.
Rezaie-Majd A, Maca T, Bucek RA, Valent P, Muller MR, Husslein P, Kashanipour A, Minar E, Baghestanian M. Simvastatin reduces expression of cytokines interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 in circulating monocytes from hypercholesterolemic patients. Arterioscler Thromb Vasc Biol. 2002; 22: 1194–1199.
Ridker PM, Rifai N, Pfeffer MA, Sacks F, Braunwald E; the Cholesterol and Recurrent Events (CARE) Investigators. Long-term effects of pravastatin on plasma concentration of C-reactive protein. Circulation. 1999; 100: 230–235.
Fukumoto Y, Libby P, Rabkin E, Hill CC, Enomoto M, Hirouchi Y, Shiomi M, Aikawa M. Statins alter smooth muscle cell accumulation and collagen content in established atheroma of watanabe heritable hyperlipidemic rabbits. Circulation. 2001; 103: 993–999.
Molloy KJ, Thompson MM, Schwalbe EC, Bell PR, Naylor AR, Loftus IM. Comparison of levels of matrix metalloproteinases, tissue inhibitor of metalloproteinases, interleukins, and tissue necrosis factor in carotid endarterectomy specimens from patients on versus not on statins preoperatively. Am J Cardiol. 2004; 94: 144–146.
Sukhova GK, Williams JK, Libby P. Statins reduce inflammation in atheroma of nonhuman primates independent of effects on serum cholesterol. Arterioscler Thromb Vasc Biol. 2002; 22: 1452–1458.
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: 684–691.
Verhoeven BA, Velema E, Schoneveld AH, de Vries JP, de Bruin P, Seldenrijk CA, de Kleijn DP, Busser E, van der GY, Moll F, Pasterkamp G. Athero-express: differential atherosclerotic plaque expression of mRNA and protein in relation to cardiovascular events and patient characteristics. Rationale and design. Eur J Epidemiol. 2004; 19: 1127–1133.
North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. N Engl J Med. 1991; 325: 445–453.
Halliday A, Mansfield A, Marro J, Peto C, Peto R, Potter J, Thomas D. Prevention of disabling and fatal strokes by successful carotid endarterectomy in patients without recent neurological symptoms: randomised controlled trial. Lancet. 2004; 363: 1491–1502.
Pasterkamp G, Schoneveld AH, van der Wal AC, Haudenschild CC, Clarijs RJ, Becker AE, Hillen B, Borst C. Relation of arterial geometry to luminal narrowing and histologic markers for plaque vulnerability: the remodeling paradox. J Am Coll Cardiol. 1998; 32: 655–662.
Verheijen JH, Nieuwenbroek NM, Beekman B, Hanemaaijer R, Verspaget HW, Ronday HK, Bakker AH. Modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase. Biochem J. 1997; 323 (Pt 3): 603–609.
Vernooy JH, Lindeman JH, Jacobs JA, Hanemaaijer R, Wouters EF. Increased activity of matrix metalloproteinase-8 and matrix metalloproteinase-9 in induced sputum from patients with COPD. Chest. 2004; 126: 1802–1810.
LaRosa JC, Grundy SM, Waters DD, Shear C, Barter P, Fruchart JC, Gotto AM, Greten H, Kastelein JJ, Shepherd J, Wenger NK. Intensive lipid lowering with atorvastatin in patients with stable coronary disease. N Engl J Med. 2005; 352: 1425–1435.
Ridker PM, Cannon CP, Morrow D, Rifai N, Rose LM, McCabe CH, Pfeffer MA, Braunwald E. C-reactive protein levels and outcomes after statin therapy. N Engl J Med. 2005; 352: 20–28.
Kennedy J, Quan H, Buchan AM, Ghali WA, Feasby TE. Statins are associated with better outcomes after carotid endarterectomy in symptomatic patients. Stroke. 2005; 36: 2072–2076.
Ascer E, Bertolami MC, Venturinelli ML, Buccheri V, Souza J, Nicolau JC, Ramires JA, Serrano CV Jr. Atorvastatin reduces proinflammatory markers in hypercholesterolemic patients. Atherosclerosis. 2004; 177: 161–166.
Crisby M, Nordin-Fredriksson G, Shah PK, Yano J, Zhu J, Nilsson J. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization. Circulation. 2001; 103: 926–933.
Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.
Shiomi M, Yamada S, Ito T. Atheroma stabilizing effects of simvastatin due to depression of macrophages or lipid accumulation in the atheromatous plaques of coronary plaque-prone WHHL rabbits. Atherosclerosis. 2005; 178: 287–294.
Nissen SE, Tuzcu EM, Schoenhagen P, Brown BG, Ganz P, Vogel RA, Crowe T, Howard G, Cooper CJ, Brodie B, Grines CL, DeMaria AN. Effect of intensive compared with moderate lipid-lowering therapy on progression of coronary atherosclerosis: a randomized controlled trial. JAMA. 2004; 291: 1071–1080.
Kowalski J, Okopien B, Madej A, Zielinski M, Belowski D, Kalina Z, Herman ZS. Effects of atorvastatin, simvastatin, and fenofibrate therapy on monocyte chemoattractant protein-1 secretion in patients with hyperlipidemia. Eur J Clin Pharmacol. 2003; 59: 189–193.
Hartung D, Sarai M, Petrov A, Kolodgie F, Narula N, Verjans J, Virmani R, Reutelingsperger C, Hofstra L, Narula J. Resolution of apoptosis in atherosclerotic plaque by dietary modification and statin therapy. J Nucl Med. 2005; 46: 2051–2056.
Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR, Braunwald E; Cholesterol and Recurrent Events Trial investigators. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med. 1996; 335: 1001–1009.
Sever PS, Poulter NR, Dahlof B, Wedel H. Different time course for prevention of coronary and stroke events by atorvastatin in the Anglo-Scandinavian Cardiac Outcomes Trial-Lipid-Lowering Arm (ASCOT-LLA). Am J Cardiol. 2005; 96: 39F–44F.