Suppression of Rage as a Basis of Simvastatin-Dependent Plaque Stabilization in Type 2 Diabetes

【摘要】  Objective- Receptor for advanced glycation end products (AGEs) (RAGE) plays a central role in the process of plaque rupture in diabetic patients. Recently, it has been reported that RAGE may be downregulated by improving glycemic control. In contrast, despite being well known that RAGE may be induced in human vessels in a glucose-independent fashion, also by myeloperoxidase (MPO)-dependent AGE generation, no data exist regarding the possibility of a pharmacological modulation of glucose-independent RAGE generation. Thus, the aim of this study was to characterize the effect of simvastatin on the expression of RAGE and RAGE-dependent plaque-destabilizing genes in human atherosclerotic plaques.

Methods and Results- Seventy type 2 diabetic patients 70%) were randomized to American Heart Association (AHA) step 1 diet plus simvastatin (40 mg/d) or AHA step 1 diet alone for 4 months before endarterectomy. Plaque expression of MPO, AGEs, RAGE, NF- B, COX-2, mPGES-1, matrix metalloproteinase (MMP)-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, procollagen 1, and interstitial collagen was analyzed by immunohistochemistry and Western blot; zymography was used to detect MMP activity. Plaques from the simvastatin group had less ( P <0.0001) immunoreactivity for MPO, AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, and MMP-9, lipids and oxLDL; reduced ( P <0.0001) gelatinolytic activity; increased ( P <0.0001) procollagen 1 and collagen content; and fewer ( P <0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells. Of interest, RAGE inhibition by simvastatin, observed not only in plaque sections but also in plaque-derived macrophages, was reverted by addition of AGEs in vitro.

Conclusions- This study supports the hypothesis that simvastatin inhibits plaque RAGE expression by decreasing MPO-dependent AGE generation. This effect in turn might contribute to plaque stabilization by inhibiting the biosynthesis of PGE 2 -dependent MMPs, responsible for plaque rupture.

The aim of this study was to characterize the effect of simvastatin on the expression of RAGE and RAGE-dependent plaque-destabilizing genes in human atherosclerotic plaques. We found that simvastatin inhibits macrophage RAGE expression by decreasing myeloperoxidase-dependent AGE generation. This effect in turn might contribute to plaque stabilization by inhibiting the release of PGE 2 -dependent MMPs responsible for plaque rupture.

【关键词】  diabetes mellitus metalloproteinases myeloperoxidase RAGE statins

Introduction

Diabetes is associated with severe atherosclerosis in humans and represents a leading cause of morbility and mortality. 1 Chronic perturbation of diabetic vasculature leads to increased number, size, and complexity of atherosclerotic plaques. Furthermore, lesion instability is enhanced in diabetes and mediates increased incidence and severity of clinical events. 2

In the recent years, there is increasing evidence that in diabetic patients, RAGE (receptor for advanced glycation end products ) plays a central role in the cascade of events that result in accelerated atherosclerotic plaque formation, as well as plaque erosion and fissuring. In fact, it has been recently demonstrated that administration of the extracellular ligand-binding domain of the receptor, soluble (s) RAGE, to apolipoprotein (apo) E-null mice with the diagnosis of hyperglycemia suppressed both accelerated development of atherosclerotic plaques 3 and progression of established lesions toward complexity. 4,5 Furthermore, we recently extended these observations to humans by demonstrating that RAGE may promote the evolution of atherosclerotic plaques toward instability by inducing PGE 2 -dependent metalloproteinase (MMP)-2 and MMP-9 production. 6

Worth noting is the observation that the pro-atherogenic role of RAGE may be modulated in vivo. In fact, we have demonstrated that RAGE may be downregulated by improving glycemic control. 6 Furthermore, Marx et al 7 have recently showed that thiazolidinediones (TZD), insulin-sensitizer drugs, can inhibit the RAGE pathway in humans. However, no data still exist regarding the possibility that drugs affecting myeloperoxidase (MPO) may reduce the RAGE-dependent plaque instability despite it being well known that enhanced deposition of AGEs (the RAGE ligands) occurs in diabetic blood vessels driven by MPO activity in macrophages, 8 and the fact that it may upregulate RAGE expression after ligation, the latter realizing a positive loop of auto-amplification of this inflammatory reaction. 9,10 Thus, it is time to hypothesize that pharmacological inhibition of MPO in the plaque environment may turn off RAGE expression and related pro-inflammatory mechanisms, thereby slowing down atherosclerotic plaque instability.

In this light, recent clinical trials have shown that statins strongly reduce cardiovascular events and mortality in diabetics. 11,12 Clinical benefits of statins are greater than those expected on the basis of only modest change in arterial stenosis severity. 13 These clinical data suggest that statins may somehow stabilize plaques against disruption, and in fact we 14 and others 15 have recently demonstrated that statins may decrease MMP activity and increase collagen content in human plaques. However, the specific molecular mechanism(s) by which statins may influence MMP generation in diabetic plaques has not yet been completely elucidated.

The possibility that suppression of RAGE by statins might represent a mechanism of glucose-independent plaque stabilization in diabetics led us to investigate whether it would modulate MMP production and extracellular matrix metabolism in atherosclerotic plaques. Here, we reported reduced RAGE expression in carotid plaques of diabetic patients randomized to simvastatin, possibly caused by reduction in AGE generation resulting from the suppression of MPO activity.

Methods

For detailed methods, please see the online Materials and Methods, available at http://atvb.ahajournals.org

Patients

We studied 70 of 122 consecutive, not previously examined or included in clinical trials, type 2 diabetic surgical inpatients (37 mol/L, 33F; 69±4years), enlisted to undergo carotid 70%) internal carotid artery stenosis. All patients were "asymptomatic" according to North American Symptomatic Carotid Endarterectomy Trial (NASCET) classification, 16 and had low-density lipoprotein (LDL) cholesterol ranging between 70 mg/dL and 100 mg/dL. All the patients had fair glycemic control, as reflected by glycohemoglobin (HbA1c) <8%. Study patients were representative of those commonly admitted to our Vascular Surgery Unit for extracranial internal carotid artery stenosis. Patients taking chronic statin therapy in the last two months were excluded from the study. Eligible patients were randomized to 4-month treatment with American Heart Association (AHA) step 1 diet plus simvastatin (Sinvacor, Merck Sharp & Dohme) 40 mg/d or AHA step 1 diet alone. Finally, after the treatment period all patients underwent endarterectomy. Lipid profile was measured at baseline and immediately before endarterectomy. Percentage of carotid diameter reduction, procedural methods, risk factors, and concomitant therapy did not differ between the two groups ( Table ). In particular, by the time of surgery, all patients were taking 100 mg daily of aspirin. The study was approved by local ethics review committees. Written informed consent was obtained from all patients before each examination.

Characteristics of Study Patients

Immunohistochemistry

After the surgical procedure, samples were immediately frozen in isopentane and cooled in liquid nitrogen (LN 2 ). Serial sections were prepared and immunohistochemistry to detect CD68 macrophages, CD3 lymphocytes, HLA-DR, myeloperoxidase, AGEs, RAGE, p65, COX-2, mPGES-1, type 1 procollagen, oxLDL, MMP-2, and MMP-9 was performed as previously described. 17

Western Blot

Tissue extracts were subjected to western blot analysis, as previously described. 17 The antibodies for MPO, AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, and MMP-9 were used.

Zymography

Zymographic analyses on 7.5% acrylamide gel containing 0.1% gelatin on plaque homogenates or plaque-derived macrophages was performed as previously described. 17

Oil Red O Staining for Lipid Content

Two parallel sections from each plaque specimen were incubated in isopropanol, then in Oil Red O (Carlo Erba, Milan, Italy) solution and rinsed in water. One of the sections was counterstained with hematoxylin.

Sirius Red Staining for Collagen Content

Tissue sections were rinsed with distillated water, incubated with Sirius red (Sigma) in saturated picric acid and collagen content was detected as previously described. 14

EIA Determinations

Plaque content of PGE 2 were measured by using a commercially available enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, Mich), according to the manufacturer?s instructions.

NF- B Activity

Nuclear extracts from plaque specimens from diet- or statin-treated patients were obtained as described by Ohlsson et al. 18 Subsequently, to study NF- B DNA binding activity in the 2 groups of plaques, chemiluminescent EMSA was performed on a biotin-labeled oligonucleotide containing an NF- B DNA-binding consensus sequence using the Lightshift EMSA kit (Pierce), 19 according to the manufacturer?s protocols. In addition, we also analyzed the expression of activated p50 subunit by specific Trans-AM NF- B p50 transcription factor assay kit (Active Motif, Rixensart, Belgium). 6,20

Assessment of Myeloperoxidase Activity

Myeloperoxidase activity was measured as described by Sehirli et al. 21

Extraction of Macrophages From Atherosclerotic Plaques

Macrophages were selectively extracted from plaques as described by de Vries et al. 22 Then, immunocytochemistry, Western blot, and zymography were performed as illustrated. Results are representative of 3 different experiments.

Isolation and Culture of Blood Monocytes

Peripheral blood monocytes were purified and cultured from 5 healthy blood donors as previously described. 17 Control or stimulated (lipopolysaccharide, 1 µg/mL; IL-1ß, 10 ng/mL) monocytes (5 x 10 6 cells/4 mL RPMI 1640) were incubated for 48 hours at 37°C in the presence or absence of 10 µmol/L simvastatin (Merck Sharp & Dohme). AGEs (800 µg/mL) were also added to some of the cultures. AGEs were prepared as described by Basta et al 10 MMP-2 and MMP-9 activity and expression were analyzed in collected media by zymography and in scraped monocytes by Western blot respectively. The results are representative of 3 experiments using cells from different donors.

Statistical Analysis

For clinical data and histological examination, variables were compared by application of 2 test. The significance of difference in biochemical marker expression and inflammatory cell infiltration between diet-treated and simvastatin-treated patients was analyzed by Student t test. The strength of the association between plasma HbA1c and plaque RAGE expression after simvastatin treatment was assessed by linear regression analysis. Data are expressed as percentage or mean±SD. All calculations were performed using the SPSS 12.0 computer program.

Results

Clinical Results

Percentages of carotid diameter reduction after treatment did not differ between the 2 groups (-1.3±0.8% versus -1±0.9%). Baseline lipid levels were similar in the 2 groups ( Table ). At the end of the study, total cholesterol and LDL cholesterol were significantly reduced in patients treated with simvastatin (-28% and -21%, respectively), whereas they did not change in patients randomized to diet alone. High-density lipoprotein cholesterol did not change significantly in the 2 groups of patients after treatment period (+4% in the simvastatin group). Baseline HbA1c was similar in the 2 groups (7.8±0.2% versus 7.7±0.2%), and unchanged after 4 months of treatment with simvastatin (7.7±0.2% versus 7.7±0.3%). No patient in either group developed any clinical event during the study.

Inflammatory Infiltration

Immunocytochemistry revealed inflammatory infiltration in all specimens examined, more evident in the shoulder of diet-treated plaques. Plaque area occupied by macrophages and T cells was significantly greater ( P <0.0001) in diet-treated rather than in simvastatin-treated plaques ( Table; supplemental Figure I, available online at http://atvb.ahajournals.org). Inflammatory cells in plaques from diet group were always characterized by strong expression of HLA-DR, a selective marker of cell activation, which contrasted markedly with the low expression of HLA-DR in plaques from simvastatin group.

Myeloperoxidase Expression and Activity Is Reduced by Simvastatin in Diabetic Plaques

Expression of MPO and its bioproducts AGEs was markedly lower in the diabetic plaques treated with simvastatin as compared with plaques treated with diet alone (23±3% versus 7±1%, and 29±4% versus 12±3%, respectively, n=35, mean±SD; P <0.0001) ( Figure 1 ). Worth noting is the fact that the reduction in MPO expression was always associated with a comparable reduction in myeloperoxidase activity in atherosclerotic plaques from patients treated with simvastatin (8.75±0.9 versus 3.1±0.5 U/g tissue). MPO staining was observed mainly in inflammatory cells infiltrating the shoulder of the plaques.

Figure 1. Immunohistochemical analysis ( x 10) on carotid plaque sections from patients treated for 4 months with diet or simvastatin, performed by using antibodies for myeloperoxidase and AGEs. Similar regions of the plaque are showed. At higher magnification ( x 40) (insert), a prevalent AGEs localization in macrophages is apparent. These results are typical of 35 diet-treated and 35 simvastatin-treated plaques.

RAGE Expression Is Reduced by Simvastatin in Diabetic Plaques

After ligation with RAGE, AGE may upregulate RAGE expression, thus realizing a positive loop of auto-amplification of this inflammatory reaction. So, we investigated the association between AGE suppression by simvastatin and RAGE expression in diabetic plaques. Immunohistochemistry revealed strong RAGE immunoreactivity in diabetic plaques ( Figure 2 A), significantly more abundant in lesions treated with diet alone than in those treated with simvastatin (30±4% versus 9±3%, n=35, mean±SD; P <0.0001). RAGE areas characterized as macrophage-rich were localized prominently in the shoulder region, in the periphery of the lipid core and in the proximity of vasa vasorum. In fact, RAGE staining at high magnification indicated its localization in activated macrophages.

Figure 2. Immunohistochemical staining ( x 10) for RAGE on plaque sections from simvastatin- and diet-treated patients. At higher magnification ( x 40) (insert), a prevalent RAGE localization in macrophages is apparent (A). Western blot for RAGE in plaque homogenates from diet- or simvastatin-treated diabetic patients. Western blot for ß-actin is also shown as a protein loading control (B). These results are typical of 35 diet-treated and 35 simvastatin-treated plaques.

RAGE Suppression by Simvastatin Is a Glucose-Independent Phenomenon

Western blot analysis confirmed RAGE expression in plaques ( Figure 2 B), markedly higher in diabetic plaques treated with diet alone (8433±132 versus 1437±126 densitometric units (DU), mean±SD, n=35; P <0.0001). Notably, RAGE reduction in plaques treated with simvastatin was completely unrelated to glycemic control, as reflected by the absence of any correlation ( R 2 =0.08, P =NS) between plasma HbA1c and plaque RAGE content (quantified by densitometric analysis).

The Effect of Simvastatin on Diabetic Plaques Is Mediated by NF- B Inhibition

After ligation with its ligands, RAGE may activate target genes via stimulation of NF- B, consequently we investigated the association between RAGE inhibition by simvastatin and NF- B activation in plaques. NF- B activation as reflected by the analysis of the activated form of both p50 subunit was significantly higher in diet- than in simvastatin-treated plaques (22±2 versus 9±2 pg/µg of nuclear extracts, n=35; P <0.0001; supplemental Figure IIA), and showed a strong concordance with RAGE expression. EMSA shift assays also showed that the greatest DNA binding activity was with nuclear extracts from patients treated with diet alone, whereas strongly reduced binding was detected in those treated with statin (4208±224 versus 1256±147 DU, mean±SD, P <0.001) (supplemental Figure IIB). Staining for p65a was significantly higher in plaques treated with diet alone (25±3% versus 6±2%, n=35; P <0.0001) (supplemental Figure IIC), and accumulated mainly in macrophages infiltrating the plaque shoulder.

The Expression of RAGE Target Genes Is Reduced by Simvastatin

COX-2 and mPGES-1 staining was more abundant in lesions from diet-treated patients (24±3% versus 5±1%, and 20±2% versus 4±1%, respectively, mean±SD, n=35, P <0.0001) ( Figure 3 A); furthermore, COX-2/mPGES-1 accumulated in activated macrophages and smooth muscle cells at the shoulder regions. Western blot confirmed the higher expression of COX-2/mPGES-1 in plaques from diet group as compared with plaques from simvastatin group (7544±134 versus 1876±111, and 6987±124 versus 1567±117 DU, respectively, mean±SD, n=35, P <0.0001; Figure 3 B).

Figure 3. Immunohistochemical analysis of sections of atherosclerotic carotid plaques from diet-treated or simvastatin-treated diabetic patients stained ( x 10) with specific antibodies for COX-2 and mPGES-1. Similar regions of the plaque are showed (A). Western blot for COX-2 and mPGES-1 in plaque homogenates from diet- or simvastatin-treated patients. Western blot for ß-actin is also shown as a protein loading control (B). These results are typical of 35 diet-treated and 35 simvastatin-treated plaques.

The Activity of RAGE Target Genes Is Reduced by Simvastatin

Because a direct and accurate comparison of the levels of COX-2/mPGES-1 might be limited by variable antibody affinities, we also confirmed results concerning COX-2/mPGES-1 expression by EIA measurements of bioproduct PGE 2 plaque content. In fact, PGE 2 was present in higher amounts in diet-treated plaques with respect to simvastatin-treated plaques (186±17 versus 42±4 ng/mg tissue, mean±SD, n=35; P <0.0001).

Impact of Simvastatin on the Expression and Activity of PGE 2 -Dependent MMPs in Diabetic Plaques

Immunohistochemistry revealed that positivity for MMP-2 and MMP-9 was significantly more abundant in diet-treated than in simvastatin-treated lesions (24±3% versus 6±1%, and 22±2% versus 7±2%, n=35; P <0.0001) ( Figure 4 A). These data were also confirmed by Western blot (6598±121 versus 1104±89, and 6676±78 versus 1165±67 DU, n=35, mean±SD; P <0.0001). In addition, zymography ( Figure 4 B) demonstrated that extracts from diet-treated plaques contained higher amounts of the activated form of MMP-2 and MMP-9 (2559±67 versus 543±45, and 2734±83 versus 565±67 DU, n=35, mean±SD; P <0.0001).

Figure 4. Immunostaining ( x 10) for MMP-2 and MMP-9 in plaque sections from simvastatin- and diet-treated patients (A). Zymography analysis on a gelatin-impregnated 7.5% SDS gel showing gelatinolytic activity of plaque extracts from simvastatin- and diet-treated patients. Both the zymogen and the activated forms of MMP-2 and MMP-9 are evident (B). Densitometric analysis of immunohistochemistry for type 1 procollagen in plaques from simvastatin- and diet-treated individuals (C). These results are typical of 35 diet-treated and 35 simvastatin-treated plaques.

Effect of Simvastatin on Plaque Extracellular Components

Immunohistochemistry analysis showed markedly higher content of procollagen type 1 in simvastatin-treated than in diet-treated plaques (22±3% versus 8±3%, n=35, mean±SD; P <0.0001) ( Figure 4 C). Procollagen levels were always inversely associated with myeloperoxidase and RAGE levels ( R 2 =- 0.41, P <0.0001). Similarly, Sirius Red polarization showed considerably higher content of interstitial collagen in tissue sections from simvastatin group compared with sections from diet group (21±3% versus 9±2%, n=35, mean±SD; P <0.0001).

Finally, total lipids and oxidized LDL (oxLDL) plaque content was significantly lower in plaques from simvastatin-treated patients (5±2% versus 23±6%, and 4±1% versus 24±3%, respectively, n=35, mean±SD; P <0.0001).

Colocalization of Myeloperoxidase with RAGE, COX-2/mPGES-1, and MMPs in Plaque Macrophages

In the first experiment, serial sections of diet-treated plaques were incubated with primary antibodies for CD68, MPO, RAGE, COX-2, mPGES-1, MMP-2, and MMP-9. Within the lesion, all proteins accumulated in the shoulder as well as in the periphery of the lipid core. In 2 other experiments, immunofluorescence double labeling associated the expression of MPO with RAGE, COX-2, mPGES-1, and MMPs in CD68+ macrophages both in plaque sections and in plaque-derived macrophages (supplemental Figure III). Thus, these analyses confirmed the concomitant presence of MPO, RAGE, and downstream effectors in macrophages at the vulnerable region of diabetic plaques.

Macrophages Isolated From Diet-Treated and Simvastatin-Treated Plaques Recapitulate Differences in Enzyme Expression Observed In Vivo

The lower RAGE levels observed in simvastatin-treated versus diet-treated plaques could be because of a lower relative abundance of macrophages in the former tissues ( Table ). Alternatively, reduced MPO expression by macrophages exposed to simvastatin could also account for the observed differences. To address this point, we assessed the expression of MPO, RAGE, and downstream enzymes by immunocytochemical and Western blot analyses in macrophages isolated from 5 diet-treated and 5 simvastatin-treated plaques. A lower positivity for MPO, RAGE, COX-2/mPGES-1, and MMPs was observed by immunocytochemistry in macrophages from simvastatin-treated plaques. In contrast, a 5-fold higher signal ( P <0.0001) was shown by Western blot in macrophages from diet-treated diabetic plaques (6234±145 versus 877±55 DU for MPO; 5345±123 versus 768±66 DU for RAGE; 6324±106 versus 1034±65 DU for COX-2; 5487±102 versus 733±43 DU for mPGES-1; 6756±121 versus 787±65 DU for MMP-2; 5956±154 versus 834±63 DU for MMP-9; supplemental Figure IV). We conclude that the phenotype of macrophages from plaques from simvastatin group differs from that of cells from diet-treated plaques and that the differences are maintained after plating, suggesting the occurrence of sustained inhibition signals in macrophages exposed to simvastatin in vivo.

Simvastatin Regulates RAGE Expression in Inflammatory Cells Through Inhibition of AGEs

To determine if simvastatin may suppress RAGE and related pro-inflammatory genes through MPO inhibition, we examined the effect of simvastatin on RAGE expression in blood monocytes in vitro (supplemental Figure V). Lipopolysaccharide caused an enhancement ( P <0.0001) in MPO (6975±144 versus 1432±132 DU), RAGE (6567±122 versus 1011±87 DU), COX-2 (6232±156 versus 1111±135 DU), mPGES-1 (6123±142 versus 1321±78 DU), MMP-2 (5437±159 versus 1222±89 DU), and MMP-9 (6854±165 versus 1211±121 DU) levels over those detected in control monocytes. Induction of these proteins by lipopolysaccharide was significantly ( P <0.0001) inhibited by simvastatin (1145±65, 1267±107, 1549±88, 1476±45, 1322±57, and 1332±96 DU, respectively); however, the inhibition of RAGE and related genes was completely reversed by the addition of AGEs. Similar results were also observed when IL-1ß was used as a stimulus. Inhibition of RAGE by simvastatin thus appears secondary to the reduction of AGE biosynthesis.

Discussion

We previously reported that RAGE contribute to the clinical instability of atherosclerotic plaques in diabetics by promoting plaque rupture induced by PGE 2 -dependent MMPs, key enzymes in the final step of this process. 6 Now, in the present report, we provide evidence for the critical involvement of RAGE in the process of plaque stabilization realized by simvastatin. In particular, the present findings are the first to our knowledge to: (1) demonstrate a direct inhibitory effect of simvastatin on RAGE expression in human atherosclerotic lesions; (2) show in humans the possibility of RAGE regulation in a glucose-independent fashion; and (3) associate the inhibition of RAGE with the reduction of MMP activity observed after statin therapy.

Lower expression of RAGE was found in specimens obtained from carotid lesions of diabetic patients randomized to simvastatin compared with specimens obtained from patients randomized to diet alone. Notably, the hypothesis that RAGE suppression by simvastatin may have a protective impact on plaque phenotype is also supported in this study by its correlation with the parallel reduction in MMP expression, and with the strong increment in plaque collagen content after simvastatin therapy.

In previous studies we 14 and others 15,23 reported the ability of statins to reduce atherosclerotic plaque inflammatory burden and to slow lesion evolution toward rupture. In particular, a recent work by Martín Ventura et al 23 showed that a 1-month intensive treatment with atorvastatin reduced both local and systemic inflammatory mediators in patients undergoing carotid endarterectomy, as evaluated by plaque content of macrophages, MCP-1, COX-2, and circulating levels of MCP-1 and PGE 2. However, these studies did not provide any evidence regarding involvement of RAGE in pathophysiology of statin-dependent plaque stabilization. RAGE is a transmembrane receptor that, after ligation with AGEs, may induce NF B activation, ultimately promoting COX-2/mPGES-1 induction and PGE 2 -dependent MMP biosynthesis. Thus, concomitant AGE generation and RAGE expression may be necessary for the biosynthesis of PGE 2 -dependent MMPs in the setting of atherosclerotic diabetic plaques.

Interestingly, macrophages of the shoulder region contain most of the RAGE within the lesion. This finding may have functional importance, because RAGE can regulate the expression of different genes in different cell types. In particular macrophages, not present in normal arterial tissue, produce an array of metalloproteinases, including the PGE 2 -dependent MMP-2 and MMP-9, 19 considered 2 of the most important in the process of atherosclerotic plaque rupture. Thus, we have facts that allow us to confidently hypothesize that increased COX-2/mPGES-1 induction and PGE 2 generation in plaque macrophage as consequence of RAGE overexpression may enhance the synthesis of MMPs in the same cell, possibly representing a crucial step in the pathophysiology of diabetic plaque instability. In this light, our description of an association between the strong reduction of these metalloproteinases in plaque treated with simvastatin and the inhibition of RAGE in the same cells support the hypothesis that such "RAGE-related" mechanism of MMP inhibition by simvastatin may operate in vivo in diabetic patients.

Usually, the expression of RAGE in human diabetic plaques has been strictly associated with glycemic control. In fact, it has been previously demonstrated that RAGE expression is higher in diabetic patients with poor glycemic control, 6 and that RAGE may be downregulated both by amelioration of glycemic control 6 and by insulin sensitizers. 7 Now, in this study, we provide the first demonstration, to our knowledge, in humans that RAGE expression in plaque may also be modulated in a glucose-independent manner by inhibition of macrophage myeloperoxidase. This observation appears to be of interest because it could even be potentially extended to other clinical settings related to the pro-atherogenic role of RAGE, suggesting that direct generation of AGE in plaques by macrophage myeloperoxidase may be operative and potentially relevant in the setting of all those clinical conditions, such as hypercholesterolemia 14 and hypertension 24 characterized by high infiltration of activated macrophages in atherosclerotic plaques. Furthermore, because AGE-RAGE interaction results in upregulation of RAGE, and NF- B activated by RAGE may further amplify RAGE expression in inflammatory cells by a direct, positive control on gene promoter, 25 RAGE downregulation in plaque macrophages treated with simvastatin may establish a negative auto-inhibitory autocrine and paracrine feedback loop, thus suppressing this inflammatory response leading to progressive plaque destabilization.

In this study, the hypothesis that RAGE suppression by simvastatin is largely dependent on the reduction of AGE generation by myeloperoxidase is also supported by additional in vitro experiments, and by the observation that RAGE reduction in plaques is associated with comparable reduction in AGE content. In addition, the experiments on plaque-derived macrophages clearly demonstrated that the reduction in RAGE expression after simvastatin therapy was related to the enzymatic inhibition of these cells, and not merely the consequence of the reduction in the number of macrophages infiltrating the plaques.

Usually, myeloperoxidase has been strongly and independently associated with cardiovascular disease. 26 In fact, expression of human myeloperoxidase in macrophages promoted atherosclerosis in hypercholesterolemic mice. 27 Furthermore, individuals with total or subtotal myeloperoxidase deficiency appear less likely to develop cardiovascular disease. 28 Again, individuals harboring a promoter polymorphism associated with a 2-fold reduction in myeloperoxidase expression appear cardioprotected, with markedly reduced angiographic evidence of coronary artery disease, myocardial infarction, and cardiac death. 29,30 Finally, plasma and serum levels of myeloperoxidase have been shown to strongly predict risks of subsequent myocardial infarction and death. 31,32 Now, our data may provide a new molecular mechanistic hypothesis for explaining, at least in part, the higher risk of atherothrombotic events associated with myeloperoxidase in these clinical studies.

Finally, we also believe to be of interest the fact that our results were obtained in diabetic patients with normal levels of LDL cholesterol at baseline, an observation that fully supports the results of recent clinical trials 11,12,33 and that provides further support against the existence of a LDL cholesterol threshold, below which lowering it would not reduce risk in diabetics.

In conclusion, this study proposes a solid hypothesis for addressing the missing link between statin therapy and plaque stabilization in diabetics by demonstrating the inhibition of the functional myeloperoxidase/AGE/RAGE axis in human atherosclerotic lesions after simvastatin therapy, and by providing evidence that it is associated with plaque stabilization possibly by suppression of the MMP-induced matrix degradation promoting plaque rupture. These findings are also potentially important from a practical standpoint, because they raise the interesting possibility that modification of the RAGE signaling by statins might provide a novel form of therapy for plaque stabilization of diabetic patients with atherosclerotic disease and prevention of acute ischemic syndromes.

Acknowledgments

Sources of Funding

This study was supported in part by grants from the Italian Ministry of Research (PRIN 2004), and by EC FP6 funding (LSHM-CT-2004-005033). This publication reflects only the authors? views. The Commission is not liable for any use that may be made of information herein.

Disclosures

None.

【参考文献】
  King H, Aubert R, Herman W. Global burden of diabetes, 1995-2005: prevalence, numerical estimates and projections. Diabetes Care. 1998; 21: 1414-1431.

Moreno PR, Murcia AM, Palacios IF, Leon MN, Bernardi VH, Fuster V, Fallon JT. Coronary composition and macrophage infiltration in atherectomy specimens from patients with diabetes mellitus. Circulation. 2000; 102: 2180-2184.

Park L, Raman KG, Lee KJ, Lu Y, Ferran LJ Jr., Chow WS, Stern D, Schmidt AM. Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation endproducts. Nat Med. 1998; 4: 1025-1031.

Bucciarelli LG, Wendt T, Qu W, Lu Y, Lalla E, Rong LL, Goova MT, Moser B, Kislinger T, Lee DC, Kashyap Y, Stern DM, Schmidt AM. RAGE blockade stabilizes established atherosclerosis in diabetic apolipoprotein-E-null mice. Circulation. 2002; 106: 2827-2835.

Hudson BI, Harja E, Moser B, Schmidt AM. Soluble levels of receptor for advanced glycation endproducts (sRAGE) and coronary artery disease: the next C-reactive protein? Arterioscler Thromb Vasc Biol. 2005; 25: 879-882.

Cipollone F, Iezzi A, Fazia M, Zucchelli M, Pini B, Cuccurullo C, De Cesare D, De Blasis G, Muraro R, Bei R, Chiarelli F, Schmidt AM, Cuccurullo F, Mezzetti A. The receptor RAGE as a progression factor amplifying arachidonate-dependent inflammatory and proteolytic response in human atherosclerotic plaques: role of glycemic control. Circulation. 2003; 108: 1070-1077.

Marx N, Walcher D, Ivanova N, Rautzenberg K, Jung A, Friedl R, Hombach V, de Caterina R, Basta G, Wautier MP, Wautiers JL. Thiazolidinediones reduce endothelial expression of receptors for advanced glycation end products. Diabetes. 2004; 53: 2662-2668.

Kislinger T, Fu C, Huber B, Qu W, Taguchi A, Du Yan S, Hofmann M, Yan SF, Pischetsrieder M, Stern D, Schmidt AM. N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression. J Biol Chem. 1999; 274: 31740-31749.

Kislinger T, Tanji N, Wendt T, Qu W, Lu Y, Ferran LJ Jr., Taguchi A, Olson K, Bucciarelli L, Goova M, Hofmann MA, Cataldegirmen G, D?Agati V, Pischetsrieder M, Stern DM, Schmidt AM. Receptor for advanced glycation end products mediates inflammation and enhanced expression of tissue factor in the vasculature of diabetic apolipoprotein E null mice. Arterioscler Thromb Vasc Biol. 2001; 21: 905-910.

Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C, Kislinger T, Stern DM, Schmidt AM, De Caterina R. Advanced glycation end products activate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses. Circulation. 2002; 105: 816-822.

Colhoun HM, Betteridge DJ, Durrington PN, Hitman GA, Neil HA, Livingstone SJ, Thomason MJ, Mackness MI, Charlton-Menys V, Fuller JH. CARDS investigators. Primary prevention of cardiovascular disease with atorvastatin in type 2 diabetes in the Collaborative Atorvastatin Diabetes Study (CARDS): multicentre randomised placebo-controlled trial. Lancet. 2004; 364: 685-696.

MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20536 high-risk individuals: a randomized placebo-controlled trial. Lancet. 2002; 360: 7-22.

Nissen SE, Tuzcu EM, Schoenhagen P, Brown BG, Ganz P, Vogel RA, Crowe T, Howard G, Cooper CJ, Brodie B, Grines CL, DeMaria AN. REVERSAL Investigators. Effect of intensive compared with moderate lipid-lowering therapy on progression of coronary atherosclerosis: a randomized controlled trial. JAMA. 2004; 291: 1071-1080.

Cipollone F, Fazia M, Iezzi A, Zucchelli M, Pini B, De Cesare D, Ucchino S, Spigonardo F, Bajocchi G, Bei R, Muraro R, Artese L, Piattelli A, Chiarelli F, Cuccurullo F, Mezzetti A. Suppression of the functionally coupled cyclooxygenase-2/prostaglandin (PG) E synthase as a basis of simvastatin-dependent plaque stabilization in humans. Circulation. 2003; 107: 1479-1485.

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.

North Am Symptomatic Carotid Endarterectomy Trial (NASCET) Steering Committee. Stroke. 1991; 22: 711-720.

Cipollone F, Prontera C, Pini B, Marini M, Fazia M, De Cesare D, Iezzi A, Ucchino S, Boccoli G, Saba V, Chiarelli F, Cuccurullo F, Mezzetti A. Overexpression of functionally coupled cyclooxygenase-2 and PG E synthase in symptomatic atherosclerotic plaques as a basis of PG E(2)-dependent plaque instability Circulation. 2001; 104: 921-927

Ohlsson BG, Englund MC, Karlsson AL, Knutsen E, Erixon C, Skribeck H, Liu Y, Bondjers G, Wiklund O. Oxidized low density lipoprotein inhibits LPS-induced binding of nuclear factor-kappaB to DNA and the subsequent expression of tumor necrosis factor-alpha and IL-1beta in macrophages. J Clin Invest. 1996; 98: 78-89.

Matata BM, Galinanes M. Peroxynitrite is an essential component of cytokines production mechanism in human monocytes through modulation of nuclear factor-kappa B DNA binding activity. J Biol Chem. 2002; 277: 2330-2335.

Kretz-Remy C, Munsch B, and Arrigo AP. NFkappa B-dependent transcriptional activation during heat shock recovery. Thermolability of the NF-kappaB.Ikappa B complex. J Biol Chem. 2001; 276: 43723-43733.

Sehirli AÕ, Sener G, Satiroglu H, Ayanoglu-Dûlger. Protective effect of N-acetylcisteine on renal ischemia/reperfusion injury in the rat. J Nephrol. 2003; 16: 75-80.

de Vries HE, Buchner B, van Berkel TJ, Kuiper J. Specific interaction of oxidized low-density lipoprotein with macrophage-derived foam cells isolated from rabbit atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 1999; 19: 638-645.

Martin-Ventura JL, Blanco-Colio LM, Gomez-Hernandez A, Munoz-Garcia B, Vega M, Serrano J, Ortega L, Hernandez G, Tunon J, Egido J. Intensive treatment with atorvastatin reduces inflammation in mononuclear cells and human atherosclerotic lesions in one month. Stroke. 2005; 36: 1796-1800.

Cipollone F, Fazia M, Iezzi A, Pini B, Cuccurullo C, Zucchelli M, de Cesare D, Ucchino S, Spigonardo F, De Luca M, Muraro R, Bei R, Bucci M, Cuccurullo F, Mezzetti A. Blockade of the angiotensin II type 1 receptor stabilizes atherosclerotic plaques in humans by inhibiting PG E2-dependent matrix metalloproteinase activity. Circulation. 2004; 109: 1482-1488.

Li J, Schmidt AM. Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation endproducts. J Biol Chem. 1997; 272: 16498-16506.

Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. Arterioscler Thromb Vasc Biol. 2005; 25: 1102-1111.

Mcmillen TS, Heinecke JW, LeBoeuf RC. Expression of human myeloperoxidase by macrophages promotes atherosclerosis in mice. Circulation. 2005; 111: 2798-2804.

Kutter D, Devaquet P, Vanderstocken G, Paulus JM, Marchal V, Gothot A. Consequence of total and subtotal myeloperoxidae deficiency: risk or benefit? Acta Haematol. 2000; 104: 10-15.

Asselbergs FW, Tervaert JW, Tio RA. Prognostic value of myeloperoxidase in patients with chest pain. N Engl J Med. 2004; 350: 516-518.

Asselbergs FW, Reynolds WF, Cohen-Tervaert JW, Jessurun GA, Tio RA. Myeloperoxidase polymorphism related to cardiovascular events in coronary artery disease. Am J Med. 2004; 116: 429-430.

Brennan ML, Penn MS, Van Lente F, Nambi V, Shishehbor MH, Aviles RJ, Goormastic M, Pepoy ML, McErlean ES, Topol EJ, Nissen SE, Hazen SL. Prognostic value of myeloperoxidase in patients with chest pain. N Engl J Med. 2003; 349: 1595-1604.

Baldus S, Heeschen C, Meinertz T, Zeiher AM, Eiserich JP, Munzel T, Simoons ML, Hamm CW. CAPTURE Investigators. Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation. 2003; 108: 1440-1445.

LaRosa JC, Grundy SM, Waters DD, Shear C, Barter P, Fruchart JC, Gotto AM, Greten H, Kastelein JJ, Shepherd J, Wenger NK. Treating to New Targets (TNT) Investigators. Intensive lipid lowering with atorvastatin in patients with stable coronary disease. N Engl J Med. 2005; 352: 1425-1435.

作者单位：From Atherosclerosis, Hypertension and Dyslipidemia Unit (C.C., A.I., M.L.F., D.C., A.D., R.M., S.U., F.S., F.C., F.Cuccurullo, A.M., F. Cipollone), “G.d?Annunzio“ University of Chieti-Pescara, and the Center for Aging Sciences,“G. d?Annunzio“ University Foundation, Chieti-Pe