{'fraction_non_alphanumeric': 0.0379447388342165, 'fraction_numerical': 0.025170325510976532, 'mean_word_length': 5.195193434935522, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Statin Treatment, Carotid Atherosclerotic Plaque Macrophage Infiltration and Circulating Inflammatory Markers

Backround Statin treatment is considered as first line therapy in patients with atherosclerotic disease. We evaluated the effect of pre-treatment with statins on carotid plaque infiltration by macrophages and on the circulating levels of proinflammatory cytokines in patients who underwent carotid endarterectomy. Patients and Methods One hundred fourteen patients were enrolled; 89 men and 25 women (mean age 67±8 years; range 42-83 years). Fifty three patients (46%) were on statin treatment at least 3 months before endarterectomy and 61 (54%) had never received statin treatment. The serum levels of high sensitivity C reactive protein (hsCRP), serum amyloid A (SAA), tumor necrosis factor α (TNFα), interleukin (IL)-1β and IL-6 were evaluated preoperatively. The intensity of macrophage infiltration was evaluated by immunochemistry, using the monoclonal antibody CD 68. The area of the plaque covered by macrophages was measured as a proportion of the whole plaque area, using a custom designed image tool analysis. Results Patients on statins had lower serum total cholesterol levels (172±50 vs 194±35 mg/dl, p= 0.014), lower low density cholesterol levels (103±44 vs 123±31 mg/dl, p= 0.010) and lower serum hsCRP levels (1.8 [1.1-3.4] vs 3.4 [1.3-4.9] mg/l, p= 0.03), while SAA, TNFα, IL-6 and IL-1β levels did not differ between the 2 groups. The infiltration of atherosclerotic plaque by macrophages was similar in statin treated patients and in controls (0.55±0.15% vs 0.49±0.19%, p= 0.21). Conclusion Patients on statins have similar macrophage accumulation in their carotid atherosclerotic plaques compared with patients not on statins. Inflammatory markers were also similar in both groups except for hsCRP which was significantly lower in those taking statins.


INTRODUCTION
The relationship between atherosclerosis and inflammation is well established [1][2][3]. Inflammation plays a role in plaque formation; even "early atherosclerosis", fatty streaks, consists of macrophage accumulation [4]. Inflammation also has a role in plaque destabilization and rupture [5,6] since macrophages and other inflammatory molecules are increased in symptomatic plaques [7].
The aim of this study was to evaluate the inflammatory burden in human carotid artery specimens as well as circulating inflammatory marker levels, in patients on statins and those not receiving these drugs.

PATIENTS AND METHODS
We evaluated patients who underwent carotid endarterectomy for significant (>70%) carotid bifurcation stenosis. The degree of stenosis was calculated using Digital Subtraction Angiography and the North America Symptomatic Carotid Endarterectomy Collaborators study [18] criteria of evaluation and measurement of carotid artery stenosis. The criteria for patient selection have been described [19].
All patients underwent a computerized tomographic brain study and were examined by the same neurologists. Based on data from history and clinical examination the patients were characterized as symptomatic or asymptomatic. Patients with amaurosis fugax, transient ischemic cerebral symptoms or ipsilateral ischemic stroke were characterized as symptomatic. The remaining patients were characterized as asymptomatic.
The study protocol was approved by the Red Cross Hospital Ethical Committee. Informed consent was obtained from all patients.

LABORATORY ANALYSIS
Blood samples were obtained from all patients the day before endarterectomy, after an overnight fast. The samples were centrifuged for 15 min at approximately 1000 g. Serum was removed, aliquoted immediately and stored at -70° C until tested. Part of the blood sample was used to measure common serum parameters and the lipid profile. Serum interleukin (IL)-1 , IL-6 and tumor necrosis factor-(TNF ) concentrations were measured using quantitative sandwich enzyme immunoassay kits (Quantikine HS, R&D Systems, Minneapolis, USA). The lower limit of detection was <0.1 pg/ml for IL-1 , 0.04 pg/ml for IL-6 and 0.12 pg/ml for TNF ; high sensitivity C-reactive protein (hsCRP) and serum amyloid A (SAA) were assayed by high sensitivity particle enhanced immunonephelometry (N Latex, Date-Behring Marburg GmbH, Marburg, Germany). The lower limit of detection was 0.18 mg/l for hsCRP and 0.8 mg/l for SAA. Values below the lower detection limit were considered as equal with this limit in the statistical analysis; there were no missing values. The coefficient variations for the assays used were for IL-1 , IL-6, TNF , hsCRP and SAA 14%, 11%, 22%, 6% and 12%, respectively. Homocysteine levels were determined using an enzymatic assay for the Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN 46250, USA), as previously described [20].

CAROTID ENDARTERECTOMY
All carotid endarterectomy procedures were performed by 1 group of vascular surgeons at the Department of Vascular Surgery of the Red Cross Hospital in Athens, from June 2004 to May 2006. Immediately after carotid endarterectomy the specimen was transferred in 0.9% normal saline solution to the laboratory. Transverse sections 4 mm thick were prepared from each atherosclerotic lesion. The section containing the most stenotic part of the bifurcation was labelled 0, while the consecutive parts distally were labelled +1, +2, +3 etc and proximally -1, -2, -3 etc. Each part was embedded in a different paraffin block. Sequential transverse sections 4 μm apart were obtained from each block and stained with haematoxylin and eosin. These sections were used for classification of the atherosclerotic plaque according to the American Heart Association (AHA) criteria [4].

IMMUNOHISTOCHEMISTRY
In order to measure macrophage infiltration of atherosclerotic plaques, we used monoclonal antibodies to a cell marker (CD68, Q-Bend Clone, Dako Corporation, Caprinteria, California, USA). Sequential sections 4 mm apart were evaluated from the atherosclerotic part of each specimen. The number of slides evaluated per patient was 4.1±2.4 (mean±SD). The whole plaque area, excluding parts of the specimen without obvious atherosclerotic lesions, was evaluated at x400 magnification. Evaluation of the images was performed with the Image Tool analysis system (Image Pro Plus, Media Software, Cybernetics, Canada) by 2 independent examiners blinded to the clinical history, and the mean value was recorded. The results were expressed as the proportion of the whole plaque area stained with the monoclonal antibody CD 68.

STATISTICAL ANALYSIS
Continuous parameters are reported as mean ± standard deviation, non parametric variables as median (interquartile interval) and categorical variables as percentages. The t-test or Mann-Whitney test was used for comparing continuous variables between 2 groups, as appropriate. Categorical variables were compared by the chi-square test or Fisher's exact test. The Spearman correlation test was used to assess relationships between variables. A p<0.05 was considered significant.

RESULTS
Clinical and demographic characteristics of the study population are shown in Table 1. Statin treated patients had lower serum total cholesterol (176±49 vs 202±40 mg/dl, p= 0.011) and low density lipoprotein cholesterol (LDL-C 103±46 vs 127±34 mg/dl, p<0.001) than patients not receiving these drugs. The 2 groups had no differences in AHA plaque classification and in procedural complications (data not shown).
hsCRP serum levels were significantly lower in patients on statins than in the controls ( Table 2). The serum levels of SAA, TNF , IL-1 and IL-6 did not differ between the 2 study groups ( Table 2).
Patients on statin treatment had no differences in the lesion area covered by macrophages compared with patients not receiving these drugs (0.55±0.15% vs 0.49±0.19%, p= 0.21, data not shown). The interobeserver variability was moderate ( = 0.61±0.09).

DISCUSSION
The main finding of this study is that patients on statins had the same degree of macrophage infiltration of their carotid atherosclerotic plaque with patients not receiving these drugs although serum hsCRP levels were significantly lower.
Several, but not all, studies favour the hypothesis that statins have beneficial effect on macrophage accumulation. Tahara et al. [21] using fluorodeoxyglucose positron emission tomography reported that simvastatin reduces the number of inflammation-rich atherosclerotic plaques compared with a placebo group. Crisby et al. [22] reported that patients pre-treated with pravastatin had less inflammation in their carotid arteries, compared with patients on placebo. Similar results were reported in monkeys, using simvastatin or pravastatin [23]. On the other hand, Verhoeven et al. [24] demonstrated that statin treated patients have increased amount of macrophages in their carotid endarterectomy specimens. However, in patients treated with atorvastatin, the increased amount of CD68 positive cells was not associated with increased protease activity [24]. Our findings are not consistent with either of these hypotheses, since we did not demonstrate any difference in macrophage infiltration between patients receiving statins and those not on these drugs.
hsCRP was lower in patients treated with statins, while the levels of SAA, TNF , IL-1 and IL-6 were not different between the 2 groups. Statin-induced reduction in hsCRP levels have been demonstrated in many studies in different patient populations [25][26][27][28]. The same is also true for SAA [29] although we did not observe this effect.

STUDY LIMITATIONS
This was a prospective cross sectional study comparing patients receiving statins for more than 3 months with patients who never received these drugs. The different statins, with varying dosage and treatment duration is a limitation. The possible effect of other drugs (like angiotensin converting enzyme inhibitors, angiotensin receptor blockers, betablockers and others) on macrophage accumulation also needs to be considered. The antibody CD68 used to validate macrophages is a non-selective macrophage marker and cannot express the complicated functional status of the macrophages [24]. We also do not know if more aggressive LDL-C lowering would change the results.

CONCLUSIONS
Patients on statins have no differences in macrophage accumulation in carotid atherosclerotic plaques compared with patients who never took these drugs. Inflammatory markers were similar in both groups except for hsCRP which was significantly lower in those individuals taking statins.