Statin therapy reduces inflammatory markers in hypercholesterolemic patients with high baseline levels

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1 722 Original Article Statin therapy reduces inflammatory markers in hypercholesterolemic patients with high baseline levels Yuki Horiuchi 1, Satoshi Hirayama 1, Satoshi Soda 2, Utako Seino 3, Mika Kon 1, Tsuyoshi Ueno 1, Mayumi Idei 1, Osamu Hanyu 4, Takashi Tsuda 5, Hirotoshi Ohmura 6, and Takashi Miida 1 1 Department of Clinical Laboratory Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan 2 Department of Endocrinology and Metabolism, Niigata City General Hospital, Niigata, Japan 3 Department of Clinical Nephroscience, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 4 Department of Endocrinology and Metabolism, Niigata University Medical and Dental Hospital, Niigata, Japan 5 Department of Cardiology, Kido Hospital, Niigata, Japan 6 Department of Cardiology, Juntendo University Faculty of Medicine, Tokyo, Japan Aim: Hypercholesterolemic patients with inflammation are at high risk for cardiovascular events. Statins exert anti-inflammatory action independent of their cholesterol-lowering action. This study sought to clarify whether statin therapy reduces inflammatory markers in hypercholesterolemic patients and to determine factors that predict the reduction in these markers. Methods: Fasting concentrations of lipoproteins and inflammatory markers were measured in 54 hypercholesterolemic patients, and age- and gender-matched healthy volunteers. Carotid atherosclerosis was determined by ultrasonography. Blood samples were also analyzed in hypercholesterolemic patients after 4 weeks of statin therapy. Results: The high-sensitivity C-reactive protein (hs-crp) and serum amyloid A (SAA) protein concentrations did not differ between the two groups. Statin therapy reduced the hs-crp and SAA concentrations in hypercholesterolemic patients from 0.75 to 0.60 mg/l ( p 0.05), and from 3.95 to 3.20 g/ml ( p 0.20), respectively. These reductions were significant for both markers, but only in subgroups with high baseline concentrations. Statins exhibited different results for hs-crp and SAA in the presence of carotid atherosclerosis. Conclusions: Statin therapy reduces inflammatory markers in hypercholesterolemic patients, and this anti-inflammatory action is limited to patients whose inflammatory markers are elevated at baseline. J Atheroscler Thromb, 2010; 17: Key words; Statin, Pleiotropic effect, Inflammation, hs-crp, SAA Introduction Hypercholesterolemia is one of the most important risk factors for cardiovascular diseases 1, 2). Due to advances in drug therapy over the last two decades, most hypercholesterolemic patients can now be easily managed in clinical practice. Statins inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG- Address for correspondence: Satoshi Hirayama, Department of Clinical Laboratory Medicine, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo , Japan sthiraya@juntendo.ac.jp Received: August 14, 2009 Accepted for publication: December 15, 2009 CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis, and lower the low-density lipoproteincholesterol (LDL-C) concentration by 20 45% 3). Because statins are much more effective and safer than any other cholesterol-lowering agents, they are used as a first-line drug worldwide. Furthermore, many clinical trials have demonstrated that statins significantly reduce both incipient and recurrent events of cardiovascular disease (CVD) 4-9). These favorable effects of statins are observed independent of gender, age, race, and baseline LDL-C concentration 3, 8, 9). In recent years, more attention has been paid to the role of low-grade inflammation in the initiation and development of atherosclerosis 10, 11). In general,

2 723 serum acute phase proteins are used as inflammatory markers to evaluate low-grade inflammation. Several prospective studies have revealed that hypercholesterolemic patients with low-grade inflammation are at a higher risk for CVD events than those without it 12, 13), and high-sensitivity C-reactive protein (hs-crp) is frequently measured for this purpose. Serum amyloid A (SAA) protein is another acute phase protein, produced not only in the liver, but also in extra-hepatic tissues 14). Experimental and clinical studies strongly suggest that statins have pleiotropic effects not related to their cholesterol-lowering action 3, 15, 16). Anti-inflammatory activity is one of the effects of statins; however, only a small number of studies have measured both markers in patients treated with statins. The purpose of this study was to investigate whether statin therapy reduces hs-crp and SAA concentrations in hypercholesterolemic patients, and to determine which factors predict a reduction in these markers. Subjects and Methods Subjects We examined two groups; the first consisted of 54 consecutive patients with hypercholesterolemia (total cholesterol [TC] concentration 220 mg/dl), according to the criteria of the Japan Atherosclerosis Society 17). They were originally screened from subjects receiving an annual health checkup at Kido Hospital. We excluded patients who were taking medicines for hypercholesterolemia. After determining serum lipoprotein profiles in the fasting state, statin therapy was initiated in all patients. We used pravastatin (n 13; 10 mg, 11; 5 mg, 2), simvastatin (n 9; 5 mg), fluvastatin (n 8; 30 mg), atorvastatin (n 21; 5 mg, 3; 10 mg, 18), and pitavastatin (n 3; 2 mg). The second group consisted of 54 age- and gender-matched healthy volunteers whose TC and triglyceride (TG) concentrations were less than 220 mg/dl and 150 mg/dl, respectively. We excluded subjects with acute and chronic infectious disease from both groups. Informed consent was obtained from all participants before enrollment. This study was approved by the institutional review board of Kido Hospital, and conducted in full accordance with the Helsinki Declaration. Laboratory measurements We took blood samples from each subject after at least a 12 h-fast. Blood samples were obtained again after 4 weeks of treatment in hypercholesterolemic patients receiving statins. Serum concentrations of TC and TG were measured by enzymatic methods. LDL-C and high-density lipoprotein-cholesterol (HDL-C) concentrations were measured by homogeneous methods. Apolipoprotein (Apo)A, ApoA, ApoB, ApoC, ApoC, and ApoE concentrations were determined using a commercial turbidimetric immunoassay kit. The hs-crp and SAA concentrations were determined by latex immunoturbidimetry; the detection limits of the system were 0.02 mg/l for hs-crp and 0.5 g/ml for SAA. Evaluation of carotid atherosclerosis We measured intima-media thickness (IMT) by B-mode ultrasonography, with a linear-type 7.5-MHz probe. Bilateral carotid arteries were scanned at longitudinal and transverse projections. We examined both proximal and distal walls of a 30-mm long segment of the common carotid artery, carotid bulb, and a 10-mm long internal carotid artery. The greatest IMT was defined as the maximum IMT. When the maximum IMT was equal to or more than 1.1 mm, the lesion was considered a carotid plaque. Statistical analyses Data are presented as means SD, or s (interquartiles). Student s or Welch s t-test was used to compare mean values, whereas the Mann-Whitney U-test was used to compare values between groups. For comparisons between baseline and posttreatment levels, a paired t-test or Wilcoxon s signedranks test was used. Statistical analyses were performed using the add-in software Statcel (OMS, Tokorozawa, Japan), designed for Microsoft Excel. Values of p 0.05 were deemed significant. Results Clinical characteristics of the study subjects Hypercholesterolemic patients had similar baseline clinical characteristics to the control subjects. There was no significant difference in their backgrounds, including the gender ratio, age, hypertension, prevalence of diabetes, current smokers, or regular drinkers (Table 1). TC and TG concentrations were 29.2% and 27.3% higher, respectively, in hypercholesterolemic patients than in the control group. The LDL-C concentration was 46.7% higher in hypercholestorolemic patients than in the control group. Furthermore, apoa, apob, apoc, apoc, and apoe concentrations were significantly higher in hypercholesterolemic patients than in the control group. In contrast, HDL-C, apoa, hs-crp, and SAA concentrations did not differ significantly between

3 724 Table 1. Clinical characteristics of the study subjects Control group Hypercholesterolemic group Men (n 17) Women (n 37) Whole group (n 54) Men (n 17) Women (n 37) Whole group (n 54) Age (years) BMI (kg/m 2 ) Hypertension (%) Diabetes (%) Smoker (%) Drinker (%) TC (mg/dl) TG (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) ApoA (mg/dl) ApoA (mg/dl) ApoB (mg/dl) ApoC (mg/dl) ApoC (mg/dl) ApoE (mg/dl) hs-crp (mg/l) SAA ( g/ml) % 0% 41.2% 58.8% ( ) ( ) % 2.7% 5.4% 13.5% ( ) ( ) % 1.9% 16.7% 27.8% ( ) ( ) % 6.3% 73.3% 66.7% ( ) ( ) % 8.6% 14.3% 6.3% ( ) ( ) % 7.8% 32.0% 28.6% ( ) ( ) Data are presented as the means SD, and the (25 th and 75 th percentiles) in high sensitivity c-reactive protein (hs-crp) and serum amyloid A (SAA). p 0.05, p 0.01, compared with control group (whole group). groups. Carotid plaques Carotid plaques were more prevalent and more advanced in hypercholesterolemic patients than in control subjects. The prevalence of carotid plaques was more than 10-fold higher in hypercholesterolemic patients than in the controls (66.7% vs. 5.6%, p 0.01). Men tended to have a higher prevalence of carotid plaques than women in hypercholesterolemic patients (Fig. 1). To assess risk factors for carotid atherosclerosis in hypercholesterolemic patients, we compared lipid profiles and inflammatory markers between those with carotid plaques (plaque group) and those without (normal IMT group), according to gender. Mean age in the male plaque group tended to be higher than in the male normal IMT group (p 0.15, Table 2), but did not differ between the female plaque group and normal IMT group. TG, apoc and the of hs-crp concentrations were higher in the male normal IMT group than in the male plaque group, which may be the result of the small number of subjects (n 4) in this group. ApoAI concentration was higher in the female normal IMT group than in the Fig.1. Prevalence of carotid plaques in hypercholesterolemic patients and controls Each group consisted of 17 men and 37 women. The prevalence of carotid plaques was significantly higher in hypercholesterolemic patients (H-cho group) than in control subjects. p values were compared with individual (male or female) control groups. female plaque group. Other variables, including SAA, showed no significant difference between groups in men or women.

4 725 Table 2. Risk factors, lipoproteins, and inflammatory markers in the hypercholesterolemic group according to gender and carotid atherosclerosis Age (years) BMI (kg/m 2 ) Hypertension (%) Diabetes (%) Smoker (%) Drinker (%) TC (mg/dl) TG (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) ApoA (mg/dl) ApoA (mg/dl) ApoB (mg/dl) ApoC (mg/dl) ApoC (mg/dl) ApoE (mg/dl) hs-crp (mg/l) SAA ( g/ml) Male hypercholesterolemic group Plaque ( ) (n 4) % 0% 66.7% 50.0% ( ) ( ) Plaque ( ) (n 13) % 8.3% 75.0% 71.4% * * ( ) ( ) Female hypercholesterolemic group Plaque ( ) (n 14) % 0% 23.1% 0% ( ) ( ) Plaque ( ) (n 23) % 13.6% 9.1% 8.3% ( ) ( ) Data are presented as the means SD, and the in the high sensitivity c-reactive protein (hs-crp) and serum amyloid A (SAA) protein. p 0.05, p 0.01, compared with the individual (male or female) hypercholesterolemic group without plaques. p 0.05, p 0.01, compared with the male hypercholesterolemic group with plaques. We also compared variables between men and women who had carotid plaques. The prevalence of smoking and drinking was significantly higher in the male plaque group than in the female plaque group ( p 0.01, p 0.05; Table 2). The of the max IMT in men tended to be greater than in women (1.4 mm [ mm (25 th and 75 th percentiles)] vs. 1.2 mm [ mm], p 0.06); however, both apoe and SAA concentrations were significantly higher in women than in men (p 0.01, p 0.05; Table 2). Effect of statin therapy on lipoproteins As expected, 4 weeks of statin therapy improved serum lipid and apolipoprotein profiles. The mean concentrations of TC, TG, and LDL-C were reduced by 24.2% (p 0.01), 19.9% (p 0.05), and 32.7% (p 0.01) of their baseline levels, respectively. The mean concentrations of HDL-C increased by 2.6% (p 0.05) of the baseline levels. Corresponding to these reductions, the levels of apob, apoc, apoc, and apoe also decreased by 26.4% (p 0.01), 20.8% (p 0.01), 13.1% (p 0.01), and 18.6% (p 0.01), respectively. However, apoa and apoa did not change significantly after statin therapy (Table 3). Effect of statin therapy on hs-crp and SAA First, we examined the effect of statin therapy on hs-crp and SAA concentrations in all hypercholesterolemic patients. Statin therapy reduced the hs- CRP and SAA concentrations from 0.75 mg/l ( mg/l [25 th and 75 th percentiles]) to 0.60 mg/l ( mg/l, p 0.05), and from 3.95 g/ml ( g/ml) to 3.20 g/ml ( g/ml, p 0.20), respectively. We found no significant correlation between reductions in LDL-C and change in either inflammatory marker ( hs-crp, rs 0.037; SAA, rs 0.069). Next, we compared statin-induced changes in hs- CRP and SAA concentrations between normal IMT and plaque groups of hypercholesterolemic patients. In the normal IMT group, neither the hs- CRP nor SAA concentration changed significantly. In the plaque group, hs-crp concentration changed from 0.70 to 0.60 mg/l ( p 0.25), while the

5 726 Table 3. Changes in lipoproteins and inflammatory markers after a 4-week statin treatment Male hypercholesterolemic group (n 17) Female hypercholesterolemic group (n 37) Whole hypercholesterolemic group (n 54) TC (mg/dl) TG (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) ApoA (mg/dl) ApoA (mg/dl) ApoB (mg/dl) ApoC (mg/dl) ApoC (mg/dl) ApoE (mg/dl) hs-crp (mg/l) SAA ( g/ml) ( 0.50, 0.30) ( 1.40, 1.20) ( 0.50, 0.00) ( 3.40, 1.00) ( 0.50, 0.20) ( 1.85, 1.08) Data are presented as the means SD, and the of high sensitivity c-reactive protein (hs-crp) and serum amyloid A (SAA) protein. p 0.05, p 0.01, compared between baseline and post-treatment level. Fig.2. Change in high-sensitivity C-reactive protein (hs-crp) concentrations by statin therapy, according to their baseline levels Hypercholesterolemic patients (n 54) were divided into three groups, based on the baseline level of hs-crp ( 1.0 mg/l, 1.0 to 2.0 mg/l, above 2.0 mg/l). Box plots show 10 th, 25 th, 75 th, and 90 th percentile cut points of hs-crp distribution for each study group. The and mean of hs-crp are indicated by a bold line and closed box. The p value for comparison between hs- CRP concentrations pre- and post-statin treatment is shown. SAA concentration tended to decrease, from 3.65 g/ml ( g/ml) to 3.00 g/ml ( g/ml, p 0.06). Finally, we compared changes in hs-crp and SAA concentrations, according to their baseline levels. In those with low and moderate baseline hs-crp concentrations (less than 2.0 mg/l), hs-crp concentration did not change significantly (Fig. 2). In contrast, hs-crp concentration decreased significantly in those with high baseline hs-crp levels. The hs-crp concentration was reduced from 2.70 mg/l ( mg/l) to 1.50 mg/l ( mg/l) in patients whose baseline hs-crp levels were above 2.0 mg/l ( p 0.05, Fig. 2). Similarly, SAA concentration did not change significantly in those with a low or moderate baseline SAA level, whereas it decreased significantly in those with a high baseline SAA level (Fig.3). Median SAA concentration was reduced from 9.30 g/ml ( g/ml) to 7.10 g/ml ( g/ml) in patients whose baseline SAA levels were above 8.0 g/ml ( p 0.05, Fig. 3). No significant difference in the reduction of inflammatory makers was found between the strong statin group (atorvastatin pitavastatin; n 24) and the other statin group (pravastatin simvastatin fluvastatin; n 30). For some of the study subjects (n 26), we evaluated the long-term effect of statins on inflammatory markers 6 months later. The hs-crp concentration was reduced from 2.30 mg/l ( mg/l) to 1.40 mg/l ( mg/l) in patients whose baseline hs-crp levels were greater than 1.0 mg/l (n 6, p 0.05). The SAA concentration was reduced from 9.00 g/ml ( g/ml) to 6.40 g/ml ( g/ml) in patients whose baseline

6 727 Fig.3. Change in serum amyloid A protein (SAA) concentrations following statin therapy, according to their baseline levels Hypercholesterolemic patients (n 54) were divided into three groups, based on the baseline level of serum amyloid A protein ( 4.0 g/ml, 4.0 to 8.0 g/ml, 8.0 g/ml). Box plots show 10 th, 25 th, 75 th, and 90 th percentile cut points of SAA distribution for each study group. The and mean of SAA are indicated by bold line and closed box. The p value for comparison between SAA concentrations pre- and post-statin treatment is shown. SAA levels were greater than 8.0 g/ml (n 5, p 0.07). Discussion The present study indicates that statin therapy reduces inflammatory markers in hypercholesterolemic patients, and that such anti-inflammatory activity is limited to those whose inflammatory markers are elevated above the baseline. We found that hs- CRP and SAA levels decreased by 25% and 20%, respectively, after 4-week statin treatment. Subgroup analysis revealed that both markers were significantly reduced only in those with baseline hs-crp 2.0 mg/l or SAA 8.0 g/ml (Figs. 2, 3). Previous randomized placebo-controlled trials suggest that the anti-inflammatory action of statins might enhance the reduction in cardiovascular events caused by their cholesterol-lowering action. In the Cholesterol and Recurrent Events (CARE) trial 8), postmyocardial patients were allocated to pravastatin and placebo control groups. Inflammation plus and minus subgroups were selected from both groups according to their hs-crp and SAA concentrations. Pravastatin treatment yielded about a two-fold reduction in the relative risk for recurrent myocardial infarction or coronary death in the inflammation plus subgroup, compared with the inflammation minus subgroup (54% vs. 25%) 18). It should be noted that none of the lipid parameters (TC, LDL-C, HDL-C, and TG concentrations) differed between these two subgroups. This finding agrees well with our observation that LDL-C did not correlate with either hs- CRP or SAA in statin-treated patients. In the Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial, healthy subjects with normal LDL-C ( 130 mg/dl) and high hs-crp ( 2.0 mg/l) concentrations were treated with rosuvastatin 20 mg daily for 1.9 years () 19). The CVD risk reduction (44%) was almost twice as high as that in other statin trials (around 20 30%). The hs-crp values were 4.2 and 4.3 mg/l in the rosuvastatin and placebo groups, respectively, which was 5- to 6-fold greater than in the present study. These data strongly suggest that such a great CVD risk reduction in the JUPITER trial resulted from the anti-inflammatory action of rosuvastatin. In vivo and in vitro studies have also found that statins reduced basal and IL- -induced CRP levels independent of cholesterol-lowering action in a human CRP transgenic mouse model and cultured human hepatocytes 20). These findings suggest a direct effect on CRP expression and agree well with our clinical results. Although both hs-crp and SAA are used as inflammatory markers, they sometimes show different responses to drug treatments. In our recent study, we examined hs-crp and SAA as well as carotid atherosclerosis in 39 women with well-controlled systemic lupus erythematosus (SLE) 21). All SLE patients were treated with low-dose steroid. The above parameters were compared with those in age- and gender-matched controls. We found that the maximum IMT in SLE patients was nearly double that in controls, independent of the menstruation status. The hs-crp concentration did not differ between groups, whereas the SAA concentration was significantly higher in the SLE group than in the control group. In another study, we measured hs-crp and SAA concentrations in hypercholesterolemic patients before and after a 4-week probucol treatment 22). The baseline concentrations of hs- CRP and SAA were as low in hypercholesterolemic patients as in the control group; however, probucol significantly reduced SAA, but not hs-crp. In the present study, statin treatment did not decrease hs- CRP both in the normal IMT and plaque group, whereas it tended to decrease SAA only in the plaque group. Such discrepancies may be attributable to differences in metabolic regulation by cytokines. The clinical significance of these different hs-crp and SAA responses remains to be determined in future

7 728 studies. Several limitations of the present study deserve consideration. First, we used five different statins to treat hypercholesterolemia. Although we did not find any significant difference among statins in their effects on inflammatory markers, the numbers in subgroups were small for any such analysis. In subgroup analysis, no significant differences in the anti-inflammatory effects were found between the strong statin group and the other statin group. Second, gender differences in the high prevalence of smoking and alcohol consumption in men may have affected atherosclerotic and inflammatory states in the subjects; however, no difference in the effect of statins was observed between men and women except in hs-crp (Table 3). Third, the baseline hs-crp and SAA concentrations were as low in our hypercholesterolemic patients as in control subjects (Table 1). Furthermore, the plaque group of our hypercholesterolemic subjects had only mild carotid atherosclerosis. 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