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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 11 4489-4496
Copyright © 2006 by The Endocrine Society

Direct Demonstration of an Antiinflammatory Effect of Simvastatin in Subjects with the Metabolic Syndrome

Sridevi Devaraj, Emily Chan and Ishwarlal Jialal

Laboratory for Atherosclerosis and Metabolic Research, University of California, Davis, Medical Center, and Veterans Affairs Medical Center, Sacramento, California 95817

Address all correspondence and requests for reprints to: I. Jialal, M.D., Ph.D., Director, Laboratory for Atherosclerosis and Metabolic Research, 4635 II Avenue, Res 1 Building, Room 3000, University of California, Davis, Medical Center, Sacramento, California 95817. E-mail: ishwarlal.jialal{at}ucdmc.ucdavis.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Metabolic syndrome (MS) is characterized by low-grade inflammation and confers an increased risk for cardiovascular disease. Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) reduce cardiovascular events in MS patients. There is a paucity of data examining the effect of statins on inflammation in MS.

Objective: We aimed to test the effect of simvastatin (40 mg/d) compared with placebo on biomarkers of inflammation [high-sensitivity C-reactive protein (hsCRP) and monocytic cytokines TNF, IL-6, and IL-1] in MS subjects.

Design and Patients: We conducted a randomized, double-blind, placebo-controlled study at the University of California, Davis, Medical Center.

Participants: Participants were subjects with MS.

Intervention: Simvastatin (40 mg/d) or placebo was administered for 8 wk.

Methods and Results: The hsCRP levels were assayed using a high-sensitivity immunoassay. Monocyte cytokines were assayed by ELISA after activation with lipopolysaccharide. Simvastatin therapy significantly decreased hsCRP levels in MS subjects compared with placebo (P < 0.0005) and resulted in a significant reduction in plasma and lipopolysaccharide-activated monocytic release of IL-6 and TNF (P < 0.025). Simvastatin therapy significantly decreased nuclear factor-{kappa}B and increased Akt activity in MS subjects compared with placebo. To gain mechanistic insights, human monocytes were pretreated with lovastatin with and without mevalonate or a phosphatidyl-3-kinase inhibitor or Rho kinase inhibitor. Lovastatin significantly decreased Rho kinase and nuclear factor-{kappa}B activity, significantly increased Akt activity, and resulted in decreased monocyte IL-6 levels; these effects were reversed with mevalonate and geranylgeranyl pyrophosphate, indicating direct effects of statins on protein prenylation.

Conclusions: Thus, we show a direct antiinflammatory effect of simvastatin therapy in MS. These findings could partly explain the benefit of statin therapy in these patients.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
ONE IN FOUR INDIVIDUALS in the U.S. population have the metabolic syndrome (MS) (1). Subjects with the MS have an increased 5-yr incidence and progression of carotid atherosclerosis and coronary heart disease (2, 3). The MS also predicts type 2 diabetes mellitus (4), a cardiovascular risk equivalent. Several lines of evidence point to the pivotal role of inflammation in atherogenesis (5). C-reactive protein (CRP), the prototypic marker of inflammation, is a risk marker for future cardiovascular events (5). In addition, recent lines of evidence suggest that it promotes atherothrombosis (6). Circulating CRP levels are elevated in subjects with the MS (7, 8). Monocytes are pivotal cells in all stages of atherogenesis and secrete proinflammatory cytokines such as IL-1ß, IL-6, and TNF, which in turn promote CRP synthesis. Recent studies indicate increased mononuclear cell activity in subjects with MS (9, 10). Also, the monocyte is a pivotal inflammatory cell that is crucial with respect to the adipose tissue contribution to inflammation. Monocyte-macrophages directly influence adipocyte biology and systemic insulin resistance in mice. Weisberg et al. (11) and Xu et al. (12) have shown that adipose tissue macrophages increase in obesity and that macrophage-related inflammation contributes to the pathogenesis of obesity-induced insulin resistance. Kanda et al. (13) have shown that monocyte chemoattractant protein 1 (MCP-1) expression in adipose tissue results in macrophage infiltration in the adipose tissue, resulting in insulin resistance, hepatic steatosis, and obesity in mice. Furthermore, in CC-motif chemokine receptor-2-deficient obese mice, there was reduced macrophage content and adipose tissue inflammation and improved glucose homeostasis and insulin sensitivity (14). Indeed, the percentage of macrophage infiltration in adipose tissue correlates with obesity and insulin resistance (15). Also, IL-6 levels are increased in MS, and 30% of plasma IL-6 derives from the adipose tissue (16). The reduction in cardiovascular events observed with statins appears to be a result of their beneficial effects on lipids and other pleiotropic antiinflammatory effects (17). Several recent reports have demonstrated the antiinflammatory effects of statins (17, 18, 19, 20, 21, 22, 23). However, there is a paucity of data in individuals with the MS specifically. In the Heart Protection Study, simvastatin (40 mg/d) effectively reduced cardiovascular events in both diabetic and nondiabetic subjects (24). We have previously demonstrated that CRP reduction with statins is a class effect (25). Also, statin therapy has been shown to decrease cardiovascular events in patients with MS (26). The aim of the present study is to test the effect of simvastatin (40 mg/d) on biomarkers of inflammation [high-sensitivity (hs)CRP and monocytic cytokines] in subjects with the MS. In this study, we examine both circulating as well as monocyte-derived biomarkers of inflammation to understand the contribution of monocyte activation to inflammation in the MS.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
This study was approved by the Institutional Review Board at University of California, Davis, Medical Center, and all subjects gave informed consent. This was a randomized, double blind, placebo-controlled study. For this study, subjects with the MS (n = 50) were studied. Exclusion criteria were as follows: smoking; use of lipid-lowering drugs; diabetes and/or hypertension on drug treatment; aspirin therapy; antiinflammatory drugs; infection; cancer; recent major surgery; illness; or liver, renal, or uncompensated metabolic/hormonal disorders. MS was defined using the criteria of the National Cholesterol Education Program Adult Treatment Panel III (27). Briefly, the patient needed to have at least three risk factors to sustain the diagnosis.

At the baseline visit, subjects were randomized to receive either placebo or simvastatin (40 mg/d) for a period of 8 wk. Fasting blood was obtained at baseline and at the end of an 8-wk period in each group (placebo and 40 mg/d of simvastatin) for measurement of the lipid profile, hsCRP, isolation of monocytes for cytokines, and other parameters of inflammation. All routine chemistry was conducted by the standard laboratory techniques in the Clinical Biochemistry laboratory. CRP levels were measured on two baseline samples and one sample at 8 wk of therapy using a hs assay (25). Any individual with an hsCRP at baseline of more than 10 mg/liter was excluded from this study because this is suggestive of overt inflammation.

Mononuclear cells were isolated from fasting heparinized blood (60 ml at baseline and at 8 wk) by Ficoll-Hypaque gradient as described previously (28) after which monocytes were isolated by magnetic cell sorting using the depletion technique (Miltenyi Biotech, Bergisch Gladbach, Germany) (28). Isolated monocytes were activated using lipopolysaccharide (LPS, 100 ng/ml).

The release of the cytokines IL-1ß, IL-6, and TNF-{alpha} was assayed in the plasma and supernatants of resting and LPS-activated monocytes after an overnight incubation at 37 C using a highly sensitive immunoassay as reported previously, using reagents from BioSource International (Camarillo, CA); the coefficient of variation of these assays was less than 6% (28). Cytokine secretion from monocytes was expressed as nanograms per milligram cell protein.

To examine mechanisms for the effects of simvastatin, we examined phosphatidylinositol-3 (PI3) kinase activity as well as Akt activity in monocytic lysates because it has been previously shown in endothelial cells that statins up-regulate Akt (29). We also examined nuclear factor-{kappa}B (NF{kappa}B) p65 activity in nuclear extracts. Nuclear extracts were prepared as described previously (30), and NF{kappa}B p65 activity was determined by ELISA using reagents from Biosource International. After immunoprecipitation of monocytic lysates before and after statin therapy, using the anti-PI3 kinase antibody (Upstate Biotechnology, Lake Placid, NY), PI3 kinase activity was assayed using a competitive ELISA (Echelon Biosciences, Salt Lake City, UT) in which the signal is inversely proportional to the amount of phosphatidyl inositol 3 phosphate [PI(3,4,5)P3] produced. After the PI3 kinase reactions are complete, reaction products are first mixed and incubated with a PI(3,4,5)P3 detector protein and then added to the PI(3,4,5)P3-coated microplate for competitive binding. A peroxidase-linked secondary detection reagent and colorimetric detection is used to detect PI(3,4,5)P3 detector protein binding to the plate. The colorimetric signal is inversely proportional to the amount of PI(3,4,5)P3 produced by PI3 kinase activity. Also, Akt activity was measured in the placebo and simvastatin group in monocytic lysates using reagents from StressGen Technologies (Victoria, British Columbia, Canada). The assay is based on a solid-phase ELISA that uses a synthetic peptide as a substrate for protein kinase B and a polyclonal antibody that recognizes the phosphorylated form of the substrate. To validate the assay, we used the PI3 kinase inhibitor LY294002. The intraassay coefficient of variation of both assays was less than 10%.

To gain mechanistic insights into the effects of statin therapy on human monocytic cytokines, we conducted in vitro experiments in isolated human monocytes using a commercially available statin, lovastatin (1 µM, because previous studies have demonstrated that the plasma concentrations of statins are in the range of 0.1–1 µM after high-dose statin therapy) (31, 32) (Merck, personal communication) in the absence and presence of mevalonate (100 µM) or farnesyl pyrophosphate (FPP,10 µM) or geranylgeranyl pyrophosphate (GPP,10 µM) and examined nuclear NF{kappa}B p65 activity, IL-6 levels, and Akt activity as well as Rho kinase activity by a pull-down assay. In the in vivo study in MS subjects, there was insufficient lysate for measurement of Rho activity. Briefly, the cell lysates were incubated with Rhotekin Rho Binding Domain (Upstate Biotechnology) for 45 min. The agarose beads were collected and electrophoresed in 12% SDS-PAGE gel. Western blotting was performed with RhoA antibody (1:1000; Upstate Biotechnology). The Rho inhibitor Y27632 (10 µM) was used as a positive control.

Data are expressed as mean ± SD for parametric data and as median (25–75th percentile) for nonparametric data. Statistical analysis was performed by the General Clinical Research Center biostatistician using SAS software (SAS Institute, Inc., Cary, NC). After repeated-measures ANOVA, baseline and posttreatment differences between groups were assessed using Mann-Whitney (Monte Carlo two-tailed estimate) tests. Percent change and {delta} differences between groups were compared using Wilcoxon signed rank tests. Spearman rank correlation coefficients were computed to assess associations between variables of interest.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Baseline subject characteristics are provided in Table 1Go. Twenty-five subjects with the MS in each group (placebo and simvastatin) completed the study. There were no significant differences in characteristics between the two groups at baseline except for hsCRP levels, which were significantly increased in MS patients on placebo. There was a significant correlation between baseline CRP levels and systolic blood pressure in these subjects with MS.


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  		TABLE 1. Baseline subject characteristics

 
Simvastatin therapy, as shown previously in other studies (17, 18, 19, 20, 21, 22, 23), resulted in a significant reduction in total low-density lipoprotein (LDL) cholesterol and non-high-density lipoprotein (non-HDL) cholesterol (25, 32, and 42%, respectively; P < 0.005) compared with baseline and placebo. No significant differences were observed with regards to triglycerides and HDL cholesterol (Table 2Go). Fasting triglycerides at baseline were less than 200 mg/dl (median, 163 mg/dl in the simvastatin group).


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  		TABLE 2. Effect of simvastatin therapy on the lipid profile in subjects with the MS

 
Levels of hsCRP were significantly decreased after simvastatin therapy (36%; P < 0.005) compared with both baseline and placebo (Fig. 1Go). As we and others have previously reported, in this study also, there was no significant correlation between LDL cholesterol reduction and CRP reduction (r = 0.23; P = 0.66).


Figure 1
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  		FIG. 1. Effect of simvastatin therapy on hsCRP levels in subjects with the MS. The hsCRP levels were measured in serum at baseline and after 8 wk of placebo or simvastatin therapy in patients with MS as described in Subjects and Methods. Data are presented as median and interquartile range. *, P < 0.005 compared with baseline and placebo.

 
Plasma IL-6 levels were significantly decreased in the simvastatin group compared with baseline (P < 0.025; Table 3Go). There were no significant differences in plasma IL-1 and TNF levels in the simvastatin group compared with baseline (Table 3Go). Although there were reductions in basal levels of monocyte IL-1ß, IL-6, and TNF in the simvastatin group compared with baseline and placebo, only the decrease in IL-6 was significant (Fig. 2AGo). LPS-activated monocytic release of both IL-6 and TNF was significantly decreased after simvastatin therapy compared with baseline and placebo (P < 0.05 and P < 0.01, respectively) (Fig. 2Go, A and B). Although monocyte IL-1ß release was decreased after simvastatin therapy, the decrease was not statistically significant (for resting IL-1ß in simvastatin group, baseline was 0.92 ± 0.47 ng/mg protein vs. 0.79 ± 0.30 ng/mg protein at wk 8; for activated IL-1ß in the simvastatin group, baseline was 3.2 ± 1.8 ng/mg protein vs. 2.7 ± 1.2 ng/mg protein at wk 8; P > 0.05). There were no significant correlations between the reduction in hsCRP with statins and features of MS or reduction in IL-6.


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  		TABLE 3. Effect of simvastatin therapy on plasma cytokine levels in subjects with the MS

 

Figure 2
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  		FIG. 2. A, Effect of simvastatin therapy on resting and LPS-activated monocyte IL-6 levels in subjects with the MS. IL-6 levels were measured in supernatants of resting and LPS-activated monocytes at baseline and after 8 wk of placebo or simvastatin therapy in subjects with MS as described in Subjects and Methods. Data are presented as mean ± SD. **, P < 0.005 compared with baseline and placebo; *, P < 0.05 compared with baseline. B, Effect of simvastatin therapy on resting and LPS-activated monocyte TNF-{alpha} levels in subjects with the MS. TNF levels were measured in supernatants of resting and LPS-activated monocytes at baseline and after 8 wk of placebo or simvastatin therapy in subjects with MS as described in Subjects and Methods. Data are presented as mean ± SD. *, P < 0.005 compared with baseline. pr, Protein.

 
Because statins have previously been shown to down-regulate NF{kappa}B activity (33) and up-regulate the PI3 kinase/Akt pathway in endothelial cells (29), we examined the role of these pathways in the reduction of IL-6, the main agonist for CRP. Furthermore, the IL-6 promoter has NF{kappa}B binding elements. To gain mechanistic insights into the effects of simvastatin therapy on hsCRP and IL-6, we also examined NF{kappa}B activity in nuclear extracts and examined Akt activity in monocytic lysates from placebo and simvastatin groups and PI3 kinase activity in the monocytic cell lysates from the simvastatin group. Simvastatin therapy resulted in significant reduction in nuclear NF{kappa}B p65 activity in monocytes compared with baseline and placebo (36%; P < 0.05; Fig. 3AGo). In addition, after simvastatin therapy, monocytic Akt activity was significantly increased compared with baseline and placebo (26%; P < 0.05; Fig. 3BGo). To confirm this finding, we also examined PI3 kinase activity in the simvastatin group. Compared with baseline, simvastatin therapy also resulted in a significant increase in PI3 kinase activity in monocytes (41%; P < 0.01; Fig. 3CGo).


Figure 3
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  		FIG. 3. A, Effect of Simvastatin therapy on monocyte nuclear NF{kappa}B activity in subjects with the MS. NF{kappa}B p65 activity was assessed in nuclear extracts of monocytes at baseline and after 8 wk of placebo or simvastatin therapy in subjects with MS as described in Subjects and Methods. Data are presented as mean ± SD. *, P < 0.05 compared with baseline and placebo. B, Effect of simvastatin therapy on monocyte Akt activity in subjects with the MS. Akt activity was assessed in lysates of monocytes at baseline and after 8 wk of placebo or simvastatin therapy in subjects with MS as described in Subjects and Methods. Data are presented as mean ± SD. *, P < 0.05 compared with baseline and placebo. C, Effect of simvastatin therapy on monocyte PI3 kinase activity in subjects with the MS. PI3 kinase activity was assessed in lysates of monocytes at baseline and after 8 wk of simvastatin therapy in subjects with MS as described in Subjects and Methods. Data are presented as mean ± SD. *, P < 0.01 compared with baseline. Simva, Simvastatin; pr, protein.

 
To test whether the effects of statin were a result of its effects on protein prenylation, human monocytes were incubated with lovastatin in the absence and presence of mevalonate in vitro. Monocytic IL-6 levels were significantly decreased with lovastatin (1 µM), and this effect was reversed with mevalonate (100 µM) and GPP (10 µM) but not with FPP (10 µM) (Fig. 4AGo). To examine the effect of statin-induced inhibition of NF{kappa}B activity and statin-induced PI3 kinase activation on IL-6 levels, monocytes were incubated with lovastatin as well as the combination of lovastatin and PI3 kinase inhibitor or Rho kinase inhibitor. As shown previously, lovastatin inhibited LPS-induced IL-6, and this was partially reversed with coincubation with the PI3 kinase inhibitor (Fig. 4bGo). Also, the Rho kinase inhibitor significantly decreased IL-6 release from LPS-activated cells, and this effect was similar to the inhibition observed with lovastatin. Furthermore, nuclear NF{kappa}B activity was increased in LPS-activated monocytes, and this was inhibited with the Rho kinase inhibitor and with lovastatin or the combination (Fig. 4CGo). Although there was a trend to increased IL-6 and NF{kappa}B activity with the PI3 kinase inhibitor, it failed to completely reverse lovastatin inhibition of IL-6 and NF{kappa}B activity. In addition, coincubation of lovastatin with LY294002 resulted in decreased Akt activity compared with LPS-activated monocytes treated with lovastatin alone (Fig. 4DGo). Furthermore, the inhibition of Rho kinase with lovastatin was only partially reversed with coincubation with the PI3 kinase inhibitor LY294002 (Fig. 4EGo). Similar results were obtained with another PI3 kinase inhibitor, wortmannin (data not shown).


Figure 4
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  		FIG. 4. A, Effect of lovastatin on monocyte IL-6 levels in vitro. Human peripheral blood monocytes were preincubated with lovastatin (Lov or Lova, 1 µM) in the absence or presence of mevalonate (Mev, 100 µM) or GPP (10 µM) or FPP (10 µM) before activation with LPS, and IL-6 levels were measured in the supernatants as described in Subjects and Methods. Data are the means of four experiments presented as mean ± SD. *, P < 0.01 compared with LPS. B, Effect of lovastatin on monocyte IL-6 levels in vitro. Human peripheral blood monocytes (resting and LPS activated) were preincubated with lovastatin (1 µM) in the absence or presence of the Rho inhibitor Y27632 (10 µM) or PI3 kinase inhibitor LY294002 (10 µM), and IL-6 levels were measured in the supernatants as described in Subjects and Methods. Data are means of five experiments presented as mean ± SD. *, P < 0.01 compared with LPS. C, Effect of lovastatin on monocyte NF{kappa}B activity in vitro. Human peripheral blood monocytes (resting and LPS activated) were preincubated with lovastatin (1 µM) and/or the Rho kinase inhibitor Y27632 (10 µM) and/or PI3 kinase inhibitor LY294002, and NF{kappa}B activity was measured in the nuclear extracts as described in Subjects and Methods. Data are means of five experiments presented as mean ± SD. *, P < 0.05 compared with LPS. D, Effect of lovastatin on monocyte Akt activity in vitro. Human peripheral blood monocytes were preincubated with lovastatin (1 or 10 µM) and/or the Rho kinase inhibitor Y27632 (10 µM) or the PI3 kinase inhibitor LY294002, and Akt activity was measured in the lysates by Western blotting or the activity assay as described in Subjects and Methods. Data are means of five experiments presented as mean ± SD. *, P < 0.01 compared with C; #, P < 0.05 compared with LPS; *a, P < 0.05 compared with LPS plus lovastatin. E, Effect of lovastatin on monocyte Rho kinase activity in vitro. Human peripheral blood monocytes (LPS activated) were preincubated with lovastatin (1 µM) and/or the Rho kinase inhibitor Y27632 (10 µM) or the PI3 kinase inhibitor LY294002, and Rho kinase was measured in the lysates as described in Subjects and Methods. Data are representative of five experiments. pr, Protein.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The MS is a common disorder and, as we have reviewed previously (8), is a proinflammatory state that predisposes to premature atherosclerosis. Circulating CRP levels are elevated in MS and predict the development of diabetes and increased cardiovascular risk (2, 3, 4). Furthermore, in addition to adipose tissue inflammation, there is increased mononuclear cell activation in MS (9, 10). Several studies have demonstrated that macrophage infiltration in the adipose tissue in obesity results in increased adipose tissue inflammation (11, 12, 13, 14, 15).

The statin drugs effectively lower cholesterol levels in patients with and without coronary artery disease and are associated with a reduction in cardiovascular events in patients with and without MS (17, 18). Furthermore, the Heart Protection Study showed that in both diabetics and nondiabetics, the same dose of simvastatin, 40 mg/d, as used in the present study, resulted in significant reduction in cardiovascular events (24). Numerous studies have shown the statins lower biomarkers of inflammation in patients with coronary artery disease, diabetes, and hypercholesterolemia (17, 18, 19, 20, 21, 22, 23). There is, however, a paucity of data in individuals with the MS examining biomarkers of inflammation, such as CRP and proximal markers such as cytokines in plasma and monocytes. In this placebo-controlled study, we report that simvastatin therapy (40 mg/d) in subjects with MS, in addition to decreasing LDL cholesterol levels, has pleiotropic effects as evidenced by a decrease in levels of hsCRP and IL-6 and in monocytic release of cytokines, IL-6, and TNF. In addition, simvastatin (40 mg/d) significantly down-regulated NF{kappa}B activity and up-regulated Akt activity in monocytes from subjects with MS compared with baseline and placebo. Furthermore, in vitro studies in human monocytes incubated with lovastatin showed decreased IL-6, which was reversed by mevalonate and GPP but not FPP. Concomitantly, lovastatin decreased Rho activity and NF{kappa}B activity and up-regulated Akt activity, suggesting that the effects of statin therapy in vivo are a result of its effects on protein prenylation, most likely geranylgeranylation. Future studies will be targeted at exploring other pathways such as MAPK, STAT3, etc.

Several studies, including large prospective clinical trials, have reported no significant correlation between reduction in hsCRP levels and the reduction in LDL observed with the different statins. In monocytes, simvastatin, in vitro, decreased LPS-induced IL-6 release; however, these authors did not examine CRP or the mechanism of inhibition, and all their studies were performed in vitro (19). In hypercholesterolemic patients on aspirin therapy, a 3-month intervention with simvastatin (20–40 mg/d) significantly decreased levels of CRP, IL-6, and TNF (20), although cellular release of cytokines was not measured, and this was not a placebo-controlled study.

Waehre et al. (21) have demonstrated that expression of chemokines macrophage inhibitory protein and IL-8 and their receptors were significantly reduced in peripheral blood mononuclear cells (PBMC) of patients treated with statins for 6 months after myocardial infarction. In a subsequent study (22), simvastatin (20 mg/d) and atorvastatin (80 mg/d) in coronary artery disease patients significantly decreased IL-1 message and protein levels in PBMC. Previously, Rezaie-Majd et al. (23) showed that simvastatin therapy (20–40 mg/d for 6 wk) in hypercholesterolemic patients significantly reduced serum as well as PBMC levels of IL-6 and MCP-1. They also examined the effect of statin in vitro and showed that statins decreased cytokine mRNA and TNF-induced transcription of cytokine mRNA in human umbilical vein endothelial cells and PBMC; this effect was reversed with the addition of mevalonate, supporting our data. However, these studies discussed above did not concomitantly measure CRP levels as in the present study. We show that in subjects with MS, simvastatin therapy significantly decreased monocyte levels of cytokines, IL-6, and TNF. This is in support of a recent report (34) that demonstrates that simvastatin therapy (80 mg/d) for 4 d before LPS administration resulted in significant reduction in toll-like receptor 2 and toll-like receptor 4 expression on monocytes. Although simvastatin therapy was associated with decreased hsCRP levels as well as monocyte IL-6 levels, there was no significant correlation between the reductions in these two variables, probably because of the shorter half-life of IL-6. Although, in this study, we clearly show an antiinflammatory effect of simvastatin on monocytic cytokines, the decrease in plasma IL-6 could also be interpreted as statins also having an effect on the adipose tissue because 30% of plasma IL-6 derives from the adipose tissue (16). However, this has to be tested directly in future studies by studying adipose tissue biology after statin therapy.

NF{kappa}B is an important transcription factor involved in regulation of inflammatory processes (35). Activated NF{kappa}B has been identified in situ in human atherosclerotic plaques (35). Nuclear NF{kappa}B binding activity has been found to be increased in peripheral blood mononuclear cells in diabetes and obesity (36, 37). Statins have been shown to decrease NF{kappa}B activity (33). Atorvastatin treatment significantly decreased NF{kappa}B activation and MCP-1 and COX-2 mRNA expression in PBMC of patients undergoing carotid endarterectomy (38). In this study, we show in human monocytes, in vivo, that simvastatin therapy significantly decreased nuclear NF{kappa}B activity compared with baseline and placebo.

The protein kinase Akt serves as a multifunctional regulator of cell survival, growth, and glucose metabolism (39). Furthermore, Kureishi et al. (29) have shown that simvastatin up-regulated Akt and phosphorylation of endothelial NO synthase (eNOS) in human umbilical vein endothelial cells, and this was blocked by incubation with mevalonate or the PI3 kinase inhibitor wortmannin. Wolfrum et al. (40) have demonstrated that administration of simvastatin to rats significantly increased myocardial PI3 kinase activity and Akt and eNOS phosphorylation and reduced infarct size after ischemia reperfusion by 42%. Recently, it has been shown that low doses of statin activate Akt activity in endothelial cells (41). We make the novel observation in this study that, compared with placebo, simvastatin therapy results in significant activation of Akt activity in human monocytes in vivo. Thus, we extend the important observations with respect to NF{kappa}B down-regulation and Akt up-regulation by statins in endothelial cells to another pivotal cell, the monocyte.

To gain mechanistic insights into the effects of statin therapy, we examined the effect of lovastatin on monocyte function, in human monocytes in vitro, and showed a significant decrease in monocyte IL-6, which was reversed using mevalonic acid. By inhibiting mevalonate synthesis, statins also prevent catabolism of isoprenoid intermediates of the cholesterol biosynthetic pathway such as FPP and GPP (42), which are known to induce prenylation of proteins, such as Rho, Rac, etc. (43). In the present study, addition of GPP but not FPP mimics the effect of statin. Thus, it is most likely that statins affect geranylgeranylation and thereby inhibit IL-6. Geranylgeranylation of Rho has been shown to be involved in up-regulation of intercellular adhesion molecule 1, inhibition of peroxisome proliferator-activated receptor-{alpha}, and down-regulation of eNOS (44). Simvastatin and lovastatin have been shown to inhibit leukocyte function antigen binding, and this inhibition was partially reversed with mevalonate (45). Rho has also been shown to activate NF{kappa}B and is required for monocyte adhesion to endothelial cells. Furthermore, Rho down-regulates nitric oxide in endothelial cells. In endothelial cells, statins down-regulate Rho/Rho kinase (46). Thus, statins may reduce inflammation (IL-6, CRP) via modulation of the Rho/Rho kinase pathway (47). Induction of CRP synthesis in the liver appears to be mainly driven by IL-6 levels, and we have recently shown that CRP production in human aortic endothelial cells is induced in presence of IL-1 and IL-6 as well as by macrophage conditioned media (48). Activation of NF{kappa}B leads to increased transcription of IL-6 and CRP (49) and both have {kappa}B binding elements in their promoter. Furthermore, inhibition of Rho activation appears to inhibit NF{kappa}B activity. Interestingly, in the mouse ischemia/reperfusion model, inhibition of Rho kinase prevented the ischemia/reperfusion-induced increase in proinflammatory cytokines seen with 24-h reperfusion, suggesting a possible role for RhoA/ROCK signaling in inflammation in vivo (47). Statins block the isoprenylation and thus the membrane targeting and functional activation of Rho family members (46). In other studies, simvastatin treatment was shown to prevent pressure overload-induced cardiac hypertrophy, and this was associated with decreased RhoA activation and p27 down-regulation (46). In endothelial cells, inhibition of ROCK, the downstream effector of Rho, also prevented NF{kappa}B activity and thrombin-induced intercellular adhesion molecule 1 expression (44).

In the present study, we show that lovastatin significantly decreases NF{kappa}B activity as well as Rho activity. Also, the Rho kinase inhibitor significantly decreased IL-6 release and NF{kappa}B activity in nuclear extracts from monocytes. It is possible that a similar effect occurred in hepatocytes because they have been suggested to be the major source of plasma CRP. In fact, recently, Arnaud et al. (50) have shown that statins decrease CRP in hepatocytes in vitro via inhibition of IL-6. Also, statins up-regulated Akt activity in vitro. Previously, Patel and Corbett (51) have shown that statins inhibit LPS-induced PI3K signaling in THP-1 cells, although the difference in the cell type (THP-1 vs. human monocyte) and the dose of statin (lovastatin 1 µM vs. simvastatin 20 µM) could account for these contrasting results. Although we did not measure plasma concentrations of statin in this study (because samples were not stored within 4–6 h after ingestion of statin), it is important to point out that previous studies have shown that plasma concentrations of 0.1–1 µM of statin can be achieved with high-dose statin therapy. In addition, statins up-regulate Akt activity, and it appears that the inhibition of IL-6 in LPS-activated monocytes is via dual pathways, mainly by inhibition of Rho and NF{kappa}B and partly by activation of Akt/PI3 kinase (see schema in Fig. 5Go). Thus, using the monocyte as an index cell, we can extrapolate our data to the hepatocyte, suggesting that IL-6 inhibition from various cellular sources (monocytes, adipose tissue, etc.) results in decreased hepatic CRP secretion.


Figure 5
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  		FIG. 5. Schema depicting the effect of statin therapy on monocyte IL-6 release.

 
Thus, we demonstrate for the first time in subjects with the MS that simvastatin therapy results in significant reduction in inflammation, as evidenced by a decrease in CRP, IL-6, monocyte IL-6, and TNF compared with placebo, down-regulates NF{kappa}B activity, and up-regulates Akt activity in human monocytes. This, coupled with the in vitro observations of decreased Rho activity by lovastatin, provides direct evidence for the antiinflammatory effects of statin therapy via protein prenylation in monocytes. Future studies will examine the role of statin therapy in directly reducing adipose tissue biology with respect to inflammation, another major contributor to systemic inflammation in the MS. Because subjects with the MS have increased inflammation and dyslipidemia, after therapeutic lifestyle changes, statins could be a therapeutic option, because benefits are obtained on the lipid profile and on biomarkers of inflammation. This could have implications for reduction in cardiovascular events in this high-risk group.


   Footnotes
 
This work was supported by National Institutes of Health Grant K24 AT00596 and a Merck Medical School Grant.

An oral presentation of this work was made at the American Heart Scientific Sessions, Dallas, TX, 2005.

First Published Online September 12, 2006

Abbreviations: CRP, C-reactive protein; eNOS, endothelial NO synthase; FPP, farnesyl pyrophosphate; GPP, geranylgeranyl pyrophosphate; HDL, high-density lipoprotein; hs, high-sensitivity; LDL, low-density lipoprotein; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1; MS, metabolic syndrome; NF{kappa}B, nuclear factor-{kappa}B; PBMC, peripheral blood mononuclear cells; PI3, phosphatidylinositol-3; PI(3,4,5)P3, phosphatidyl inositol 3 phosphate.

Received February 9, 2006.

Accepted August 29, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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