Author + information
- Received September 25, 2002
- Revision received December 13, 2002
- Accepted January 24, 2003
- Published online May 7, 2003.
- Torgun Wæhre, MD*,†,* (, )
- Jan K Damås, MD, PhD*,†,
- Lars Gullestad, MD, PhD§,
- Are M Holm, MD*,
- Terje R Pedersen, MD, PhD∥,
- Kjell E Arnesen, MD¶,
- Harald Torsvik, MD, PhD§,
- Stig S Frøland, MD, PhD*,‡,
- Anne G Semb, MD, PhD∥ and
- P.ål Aukrust, MD, PhD*,‡
- ↵*Reprint requests and correspondence:
Dr. Torgun Wæhre, Research Institute of Internal Medicine, Rikshospitalet University Hospital, N-0027 Oslo, Norway.
Objectives We sought to investigate whether the activation of the chemokine network observed in patients with coronary artery disease (CAD) could be modified by treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins).
Background Chemokines and chemokine receptors are important mediators in atherogenesis, and we hypothesized that the statins could affect the chemokine network in CAD.
Methods Thirty CAD patients without previous statin therapy were randomized to receive atorvastatin (80 mg/day, n = 15) or simvastatin (20 mg/day, n = 15). Peripheral blood mononuclear cells (PBMCs) and plasma were obtained at baseline and after six months of statin therapy. Messenger ribonucleic acid (mRNA) expression of chemokines and chemokine receptors in PBMCs was analyzed by ribonuclease protection assay and real-time reverse-transcription polymerase chain reaction. Chemokines were also examined in the supernatants from unstimulated and lipopolysaccharide-stimulated PBMCs (and in plasma).
Results Our main findings were: 1) gene expression of several chemokines (i.e., macrophage inflammatory protein [MIP]-1α, MIP-1β, and interleukin [IL]-8) and chemokine receptors (i.e., CC chemokine receptor [CCR]1, CCR2, CCR4, and CCR5) was markedly increased among CAD patients compared with healthy control subjects; 2) treatment with atorvastatin and simvastatin markedly reduced the mRNA levels of some of these chemokines (i.e., MIP-1α, MIP-1β, IL-8) and receptors (i.e., CCR1 and CCR2), with the most pronounced effect in the atorvastatin group; and 3) statin therapy reduced the spontaneous release of IL-8 and MIP-1α from PBMCs in CAD patients.
Conclusions This study demonstrates a down-regulatory effect of statins on the chemokine network in CAD patients, possibly contributing to the beneficial effects of statins in this disorder.
Atherosclerosis has elements of both lipid deposition and inflammation. Hypercholesterolemia is an established risk factor for atherosclerosis and coronary artery disease (CAD), but these patients also have raised plasma levels of inflammatory cytokines, enhanced activation of circulating leukocytes, and extensive infiltration of blood-derived macrophages and T cells into atherosclerotic plaques, reflecting an important pathogenic role of inflammation in atherogenesis (1).
Several clinical trials of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have demonstrated improved prognosis of patients with CAD (2). This effect was thought to be predominantly attributed to their lipid-lowering properties, but recent studies suggest that other properties of statins such as immunomodulatory effects may also be beneficial in CAD (3). Accordingly, the reduction of cardiovascular events observed during statin therapy appears to be particularly prominent in patients with elevated C-reactive protein, suggesting anti-inflammatory effects of these agents (4).
Recruitment of leukocytes into the vascular wall is a crucial feature of all stages of atherosclerosis. Increasing evidence suggests that chemokines or chemotactic cytokines play an important role in this process, not only by directing leukocytes into the vessel wall but also by activating these cells within the atherosclerotic lesion (5). Consequently, the chemokine network could represent an important therapeutic target in this disorder.
Although there are several reports of potential anti-inflammatory properties of statins, the knowledge of the effect of these medications on chemokines and chemokine receptors is scarce, mostly based on in vitro studies and animal models (6–8). We hypothesized that the beneficial effects of statins in CAD could be related to the effects on the chemokine network. This hypothesis was investigated in an open-label, randomized study analyzing the expression of several CC and CXC chemokines and their receptors (CCR and CXCR) in peripheral blood mononuclear cells (PBMCs) isolated from CAD patients before and after six months of therapy with “high-dose” atorvastatin or “conventional-dose” simvastatin, representing two extremities of statin therapy.
Thirty patients with a previous myocardial infarction and without statin therapy were included in the study. Exclusion criteria were myocardial infarction within the last six weeks, severe concomitant disease (e.g., infections, connective tissue disease, malignancies), congestive heart failure, and the use of medications other than aspirin with known anti-inflammatory effects. None of the patients experienced mild inflammatory or infectious disorders in the last four weeks before blood sampling. Patients were randomized to simvastatin 20 mg/day (n = 15) or atorvastatin 80 mg/day (n = 15) in an open fashion, and non-fasting venous blood was collected at baseline and after six months of therapy. The demographic and clinical characteristics of the patients are shown in Table 1, demonstrating no significant differences between the treatment groups. Fifteen healthy blood donors (age 50.1 ± 6.4 years; 4 women and 11 men) without any known cardiovascular disease served as the control group. The regional ethical committee approved the study. All patients gave their written, informed consent.
Blood sampling protocol and isolation of cells
Peripheral venous blood was drawn into pyrogen-free tubes with ethylenediamide tetraacetic acid (EDTA) or heparin as anticoagulants. For plasma, the EDTA tubes were placed on melting ice, centrifuged within 15 min at 1,500 gfor 10 min at 4°C. Plasma was stored at −80°C, and the samples were thawed only once.
The PBMCs were obtained from heparinized blood by Isopaque-Ficoll (Lymphoprep; Nycomed, Oslo, Norway) gradient centrifugation. Cells were immediately stored in liquid nitrogen as pellets or cryopreserved in 10% dimethylsulfoxide (Sigma, St. Louis, Missouri), 25% fetal calf serum (Myoclone; Gibco, Paisley, U.K.), and 65% RPMI 1640 medium with 2 mmol/l l-glutamine and 25 mmol/l HEPES buffer (Gibco), as previously described (9).
Ribonuclease protection assay
Total ribonucleic acid was isolated from frozen PBMC pellets using the RNeasy Minikit (Qiagen, Hilden, Germany), subjected to DNase I treatment (RQI DNase; Promega, Madison, Wisconsin), quantified spectrophotometrically, and stored at −80°C. The ribonuclease protection assay was performed as previously described with chemokine (hCK5) and chemokine receptor (hCR5) multiprobes from Pharmigen (San Diego, California) (10).
Real-time quantitative reverse-transcription polymerase chain reaction
Total ribonucleic acid was isolated from PBMCs, as described earlier. Real-time SyBr Green or Taq-Man quantitative reverse transcription-polymerase chain reaction was performed using the ABI Prism 7700 (Applied Biosystems, Foster City, California), and sequence-specific polymerase chain reaction primers and probes for the actual genes were: CXCR1 (forward primer [FP]: 5′-CATCAAGTGCCCTCTAGCTGTTAA-3′; reverse primer [RP]: 5′GCAATGGTTTGATCTAACTGAAGG-3′), CXCR2 (FP: 5′GCCTGTCTTACTTTTCCGAAGGA-3′; RP: 5′-CCGCCAGTTTGCTGTATTGTT-3′), CXCR4 (Taq-Man probe: 5′ ACACTTCAGATAACTACACCGAGGAAATGGG-TAMRA-5′; FP: 5′-GGTTACCATGGAGGGGATCAGTA-3′; RP: 5′CAGGGTTCCTTCATGGAGTCATAG-3′). The house-keeping gene beta-actin (Applied Biosystems) was included as an endogenous control. Standard curves were run on the same plates, and the relative standard curve method was used to calculate relative gene expression.
Protein levels of interleukin (IL)-8 (sensitivity of 10 pg/ml), macrophage inflammatory protein (MIP)-1α (sensitivity of 10 pg/ml), and monocyte chemoattractant protein (MCP)-1 (sensitivity of 5 pg/ml) were determined by enzyme immunoassay (R&D Systems, Minneapolis, Minnesota).
Cell culture experiments
Cryopreserved PBMCs were thawed as described elsewhere (9). The cells were cultured with or without lipopolysaccharide from Escherichia coli/O26:B6 (Sigma; final concentration of 10 ng/ml) in RPMI 1640 medium (Gibco), supplemented with 5% human AB+ serum (Bio Whittaker, Whalkesville, Maryland) in 96-well trays (106cells/ml, 200 μl/well; Costar by Corning, Cambridge, Massachusetts). Supernatants were harvested after 20-h culturing and stored at −80°C.
The chemokine data were not normally distributed, and non-parametric tests were chosen for the statistical analyses. The Mann-Whitney Utest was used for unpaired analyses, and the Wilcoxon matched-pair test was used for paired analyses. P values <0.05 (two-sided) were considered statistically significant. However, due to multiple comparisons, particular attention should be directed toward lower p values (i.e., <0.01).
As expected, both atorvastatin and simvastatin reduced total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels in these patients during six months of therapy, with a non-significant tendency toward a greater lipid-lowering effect in the atorvastatin group (Table 2).
Chemokine and chemokine receptor gene expression in PBMCs from CAD patients and healthy control subjects
Before initiating statin therapy, the CAD patients had markedly enhanced gene expression of MIP-1α (∼4.2-fold), MIP-1β (∼5-fold), and IL-8 (∼2.3-fold), as compared with healthy individuals (Fig. 1). In contrast, regulated upon activation, normally Tcell expressed and secreted (RANTES) was equally expressed in these groups. The signals obtained for the other chemokines (e.g., MCP-1) were low in both CAD patients and controls, making them unsuitable for quantitative analyses (Fig. 2).
Concomitant with the enhanced expression of MIP-1α and MIP-1β, the gene expression of their corresponding receptors (i.e., CCR1 and CCR5) was also increased in PBMCs from CAD patients (Fig. 1). Moreover, CCR2 (i.e., the MCP-1 receptor) and CCR4 (i.e., the receptor for thymus and activation-regulated chemokine and macrophage-derived chemokine) were also up-regulated in CAD. In contrast, CXCR1 and -2 (i.e., receptors for IL-8) and CXCR4 (i.e., the receptor for stromal cell-derived factor-1α) were equally expressed in patients and controls (Fig. 1).
Effect of statin therapy on chemokine and chemokine receptor expression
Several interesting effects were observed on gene expression of chemokines and chemokine receptors after six months of statin therapy. First, the chemokines MIP-1α, MIP-1β, and IL-8 were markedly reduced in both treatment groups, with particularly suppressing effect in the atorvastatin group, in which the signals of gene expression were nearly absent after therapy (Figs. 2 and 3). ⇓Secondly, gene expression of the chemokine receptors CCR1 and CCR2 was also significantly inhibited by the statins, with a similar effect in both treatment groups (Fig. 3). In contrast, gene expression of CCR4 and CCR5, which was markedly up-regulated in the CAD group (Fig. 1), as well as CXCR1, CXCR2, CXCR4, and RANTES, which was equally expressed in CAD patients and controls, was not affected by statin therapy (data not shown).
During statin therapy, we found no significant correlation between changes in chemokines or chemokine receptors and lipid parameters in either of the treatment groups. Moreover, most patients were taking aspirin throughout the study period, and accordingly, the effects of statins were superimposed on any possible immunomodulating effect of aspirin.
Effect of in vivo statin therapy on MIP-1α, IL-8, and MCP-1 secretion from cultured, cryopreserved PBMCs
The most prominent effect of statin therapy on gene expression of chemokines was the marked reduction of IL-8, MIP-1α, and MIP-1β. Although protein secretion from cultured, cryopreserved cells is not completely equivalent to protein levels in freshly isolated cells, we next evaluated whether the down-regulating effect of statins was also present at the protein level by analyzing the spontaneous and lipopolysaccharide-stimulated release of IL-8 and MIP-1α from cryopreserved PBMCs isolated before and after therapy. According to the important role of MCP-1 in atherogenesis, this chemokine was also analyzed in PBMC supernatants. As shown in Figure 4, atorvastatin therapy for six months (n = 13) significantly reduced the spontaneous release of both IL-8 (Fig. 4A) and MIP-1α (Fig. 4B). Although the messenger ribonucleic acid (mRNA) level of MCP-1 in freshly isolated PBMCs was low (Fig. 2), these cells spontaneously released a large amount of this chemokine after culturing for 20 h. However, and in contrast to the attenuating effect of statins on IL-8 and MIP-1α, no effect of atorvastatin was seen on MCP-1 secretion (Fig. 4C). In contrast to the inhibiting effect of statins on the spontaneous release of chemokines, no effect was seen on the lipopolysaccharide-stimulated release of IL-8, MIP-1α, or MCP-1 (data not shown). Similar patterns of chemokine secretion were seen in four patients receiving simvastatin (data not shown).
Effect of statin therapy on plasma levels of chemokines
We also examined the effect of statin therapy on plasma levels of chemokines. The plasma levels of MIP-1α and IL-8 were low, just above (MIP-1α) and below (IL-8) the detection limits of the assays in both controls and CAD patients both before and after therapy, thereby being unsuitable for statistical analyses. Taking into consideration the marked differences in gene expression of these chemokines between CAD patients and controls, this may indicate that measuring plasma levels of these chemokines, at least in stable CAD patients, as in the present study, may have substantial limitations. For MCP-1, there were no differences in plasma levels before and after statin therapy (137 ± 7.1 vs. 132 ± 5.9 pg/ml in the total study population).
Several studies in animal models suggest a crucial role of chemokines in atherogenesis (11). Although caution is needed when interpreting the results from multiple comparisons, the present study demonstrates markedly elevated mRNA levels of several chemokines and chemokine receptors in PBMCs from CAD patients, further supporting a pathogenic role of increased chemokine activity in these patients. Even more importantly, we found that statin therapy inhibited the expression of some of these chemokines (i.e., IL-8, MIP-1α, and MIP-1β) and chemokine receptors (i.e., CCR1 and CCR2) at the cellular level in PBMCs, as assessed at both the protein and mRNA levels. We suggest that the down-regulating effect on the chemokine network may contribute to the observed beneficial effects of these drugs in CAD.
The involvement of chemokines and chemokine receptors in the pathogenesis of atherosclerosis has been widely investigated, particularly focusing on the effects of IL-8 and MCP-1 and their corresponding receptors. Hence, enhanced expression of these chemokines has been found in human atherosclerotic lesions, possibly mediating chemoattractant and mitogenic effects on neutrophils, T cells, and smooth muscle cells (12). Furthermore, macrophages with enhanced expression of chemokine receptors (e.g., CXCR2) have been reported in human and murine atheroma (11). Also, knockout mice lacking IL-8 and MCP-1 or their corresponding receptors (i.e., CXCR2 and CCR2) have significantly reduced progression of atherosclerosis (11). Accordingly, the down-regulatory effect of statins on the expression of the MCP-1 receptor CCR2 in circulating PBMCs in CAD patients could potentially impair atherogenesis in these patients, at least partly by inhibiting the recruitment of leukocytes into the vessel wall, even though MCP-1 expression is unaffected. Moreover, if the down-regulatory effects of statins on IL-8 expression also exist in leukocytes within the atherosclerotic plaque, it may reduce the inflammatory, matrix-degrading, and thrombotic effects of this chemokine, thereby promoting plaque stability and reducing the susceptibility for developing plaque rupture and acute coronary syndromes.
Enhanced MIP-1α and MIP-1β activation seems to be involved in the pathogenesis of several inflammatory disorders, and there are also some reports suggesting their involvement in atherogenesis. Thus, not only MCP-1 and IL-8 but also MIP-1α, MIP-1β, and RANTES are expressed in atherosclerotic plaques (12). Interestingly, MIP-1α and MIP-1β are expressed mainly in T cells co-localized in macrophage-rich areas, suggesting cross-talk between macrophages and T cells through chemokine signaling within the atherosclerotic lesion (5). Moreover, Schecter et al. (13)recently reported that MIP-1β increased tissue factor activity in vascular smooth muscle cells, suggesting that this chemokine could mediate both inflammatory and thrombotic responses within an atherosclerotic plaque. Also, studies in CCR1 knockout mice indicate the involvement of MIP-1α in the pathogenesis of allograft atherosclerosis (14). The increased expression of MIP-1α and MIP-1β and their corresponding receptors CCR1 and CCR5 in PBMCs from CAD patients, as demonstrated in the present study, further supports a possible role of these mediators in atherogenesis. Moreover, the inhibitory effect of statins on MIP-1α and its corresponding receptor CCR1, as well as on MIP-1β, could be of importance for the anti-atherogenic effects of these drugs. The pathogenic importance of these findings needs to be further evaluated in forthcoming studies.
An important question is whether the pleiotropic effects of statins are secondary to the reduction in cholesterol levels or due to other mechanisms. Statins inhibit cholesterol synthesis but also reduce the synthesis of non-steroidal products in the mevalonate pathway. In vitro studies indicate that suppression of these mediators may be responsible for at least some of the anti-inflammatory effects seen with statins (15,16). Also, nuclear factor kappa-B, required for transcription of several inflammatory cytokine and chemokine genes, is down-regulated by statins, at least partly independent of their cholesterol-lowering properties (17). In the present study, we compared the immunomodulatory effect of two extremities of statin therapy (i.e., high-dose atorvastatin and conventional-dose simvastatin) in 30 CAD patients. Although there was a tendency toward a more prominent cholesterol-lowering effect of atorvastatin, this difference did not reach statistical significance, probably because of the relatively small sample sizes, compared with studies focusing on the cholesterol-lowering effects of these medications. However, although we could not demonstrate any differences in the effect on cholesterol levels, atorvastatin had a significantly more suppressive effect on MIP-1α and MIP-1β expression than did simvastatin, suggesting that the effects of statin on chemokines are even more potent than, and at least partly unrelated to, their lipid-lowering properties. The lack of correlation between the degree of cholesterol lowering and reduction in chemokine and chemokine receptor expression further supports such a notion. Alternatively, dose-dependent and -independent differences in the immunomodulatory potencies of atorvastatin and simvastatin may explain the more pronounced inhibitory effect of atorvastatin on MIP-1α and MIP-1β levels. Additional studies are needed to elucidate these issues. Nevertheless, despite some differences, both high-dose atorvastatin and conventional-dose simvastatin had a markedly down-regulatory effect on the chemokine network, suggesting a class effect rather than a drug-specific effect.
Although statins were found to have inhibitory effects on the chemokine network, the present study also revealed limitations of the anti-inflammatory effect of these medications. First, and in contrast to previous in vitro experiments (8), the levels of MCP-1 were not affected in our study, suggesting that this important mediator in atherogenesis is not effectively suppressed during statin therapy in CAD patients. Second, CCR5 and CXCR2 (18), which are important for the arrest and migration of leukocytes through the endothelium, remained unaltered during the treatment period. Finally, although the gene expression of MIP-1α and IL-8 from PBMCs was markedly down-regulated, the effect on the protein secretion of IL-8 was rather modest, and statins had no effect on the LPS-stimulated release of these chemokines. These differences in effect of statins on different chemokines may at least partly reflect differences in the regulation of these chemokines (19,20)and suggest a potential for other immunomodulatory treatment regimens in addition to statins in CAD and other atherosclerotic disorders.
Increasing evidence suggests that the anti-inflammatory effects of statins are not only curious side effects but also may cause some of the beneficial clinical effects of statins in CAD patients (4). Herein, we show markedly down-regulatory effects of statins on the chemokine network in CAD patients. We believe that these effects on chemokines and chemokine receptors may contribute to the beneficial effects of statins in CAD patients.
☆ This study was supported by the Norwegian Research Council, Medinnova Foundation, and Eva og Gunnar Mørkveds Foundation, all in Oslo, Norway, as well as by the Inger Haldorsens Foundation, Bergen, Norway.
- coronary artery disease
- CC chemokine receptor
- CXC chemokine receptor
- monocyte chemoattractant protein
- macrophage inflammatory protein
- messenger ribonucleic acid
- peripheral blood mononuclear cells
- regulated upon activation, normally Tcell expressed and secreted
- Received September 25, 2002.
- Revision received December 13, 2002.
- Accepted January 24, 2003.
- American College of Cardiology Foundation
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