Background—The
clinical benefits of statins are attributed to changes in plaque composition
that lead to reduced metalloproteinase (MMP) activity and plaque stabilization.
However, the molecular mechanism of this effect is unclear. Recently, we demonstrated enhanced expression
of isoforms of inducible cyclooxygenase (COX) and PGE synthase (COX-2/mPGES) in
human symptomatic plaque and provided evidence that this is associated with
MMP-induced plaque rupture. The aim of this study was to characterize the
effect of simvastatin on inflammatory infiltration and the expression of
COX-2/mPGES and MMPs in human carotid plaques.
Methods and
Results—Seventy patients with symptomatic carotid artery stenosis
were randomized to the American Heart Association Step 1 diet plus simvastatin
(40 mg/d) or the American Heart Association Step 1 diet alone for 4 months
before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2,
mPGES, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen
content by immunocytochemistry, Western blot, and reverse
transcription–polymerase chain reaction, whereas zymography was used to detect
MMP activity. Immunocytochemistry was also used to identify CD68 macrophages,
CD3 T-lymphocytes, smooth muscle cells (SMCs), and HLA-DR inflammatory cells.
Plaques from the simvastatin group had fewer (P0.0001) macrophages,
T-lymphocytes, and HLA-DR cells; less (P0.0001) immunoreactivity for
COX-2/mPGES and MMPs; reduced (P0.0001) gelatinolytic activity; increased
(P0.0001) collagen content; and reduced (P0.0001) lipid and oxLDL content.
Interestingly, COX-2/mPGES inhibition by simvastatin was completely reversed by
mevalonate in vitro.
Inflammatory processes play a pivotal role in the
pathogenesis of atherosclerosis, particularly in the progression of
atherosclerotic plaque toward instability.
Rupture-prone lesions contain a large lipid core underlying a thin and
collagen-poor fibrous cap, and they usually have prominent inflammatory
infiltrates of macrophages and lymphocytes. Macrophages produce proteolytic
enzymes capable of degrading plaque constituents, including members of the
etalloproteinase (MMP) family. In particular, expression of MMP-2 and MMP-9 has
been shown within human atherosclerotic lesions and critically implicated in
plaque rupture.
Conclusions—This study demonstrates that simvastatin decreases inflammation
and inhibits COX-2/mPGES
expression in plaque macrophages, and this effect in turn may contribute to
plaque stabilization by inhibition of MMP-induced plaque rupture.
(Circulation. 2003;107:1479-1485. {This
article fails to consider the VIGOR study, or the growing literature on how
COX-2 promotes atherosclerosis}
Production of these MMPs by macrophages occurs through a
prostaglandin (PG) E2 -dependent pathway.
Signaling through this pathway involves the modulation of cyclooxygenase
(COX) and PGE synthase (PGES). Two isoforms of COX (COX-1 and COX-2) and PGES
(cPGES and mPGES) have been identified. COX-1 and cPGES are constitutively
expressed. In contrast, COX-2 and mPGES are induced in response to several
stimuli in inflammatory diseases. Consistent with the hypothesis of COX-2/mPGES
contributing to the clinical instability of atherosclerosis, we have recently
reported the overexpression of COX-2/mPGES as a pathway underlying the enhanced
release of active MMPs in symptomatic atherosclerotic plaques.
Hypercholesterolemia is a major risk factor for
atherosclerosis, and recent clinical trials have shown that statins reduce
cardiovascular events and mortality in humans. Clinical
benefits of statins are greater than those
expected on the basis of the modest change in arterial stenosis severity.
These data suggest that statins may somehow stabilize plaques against
disruption.
In this regard, it is of extreme interest to analyze the
ability of statins to reduce gelatinolytic activity. Crisby et al recently
demonstrated that statins may decrease inflammation and MMP-2 and increase
collagen content in human carotid plaques. However, the specific molecular
mechanisms by which statins may influence MMP generation in plaque macrophages
are still unknown.
The possibility that the suppression of COX-2 and mPGES by
statins might represent a mechanism of plaque stabilization led us to
investigate whether it would modulate MMP production by macrophages into
atherosclerotic plaques. Here, we report
reduced MMP production by macrophages in carotid plaques of patients randomized
to simvastatin, most likely because of a reduction in PGE2 synthesis as a
result of the suppression of COX-2/mPGES.
Methods
Patients:
We studied 70 of 128
consecutive surgical inpatients who had been enlisted to undergo carotid
endarterectomy for extracranial
highgrade (70%) internal carotid artery stenosis. All patients were symptomatic
according to North American Symptomatic Carotid Endarterectomy Trial
classification and had LDL cholesterol (LDL-C) ranging between 100 and 129
mg/dL. Patients were randomized to 4-month treatment with the American Heart
Association Step 1 diet plus simvastatin (Sinvacor, Merck Sharp & Dohme) 40
mg/d or American Heart Association Step 1 diet alone. After the treatment
period, all patients underwent endarterectomy. Fasting plasma total
cholesterol, HDL cholesterol, LDL-C, and triglyceride levels were measured at
baseline and before endarterectomy. Procedural methods, risk factors, and
concomitant therapy did not differ between the 2 groups (Table 1). By the time
of surgery, all patients were taking 100 mg of aspirin daily, a dose that does
not affect vascular COX-2 because of rapid de novo synthesis of the enzyme in
nucleated cells during the 24-hour dosing interval. The study was approved by
local ethics review committees. Written informed consent was obtained from all
patients before each examination.
Immunohistochemistry
Samples were frozen in isopentane and cooled in liquid
nitrogen. Serial sections were prepared
as described previously and incubated with the specific antibodies anti-CD68,
anti-CD3, anti-HLA-DR, anti--smooth muscle actin, and anti-CD31 (Dako
Corporation); anti-COX-1, anti-COX-2, and anti-mPGES (Cayman Chemical);
anti-MMP-2 and anti-MMP-9
(Calbiochem-Novabiochem); and anti-oxLDL (a gift from Dr Raffaella
Muraro). In addition, 4 sections from each plaque were examined for the
presence of plaque ulceration and intraplaque hemorrhage. The specimens were
analyzed by an expert pathologist (intraobserver variability 6%) blinded to the
patient’s therapy. Quantitative Analysis
for Histology CD3-positive T cells were counted individually and expressed as
the number of cells per square millimeter of section area as determined by computer-aided
planimetry (see below). Furthermore, we determined the area occupied by
CD68-positive and -actin–positive cells planimetrically and calculated the
percentage of macrophage-rich and smooth muscle cell (SMC)–rich areas. Analysis
of experiments was performed with a PC-based 24-bit color image-analysis
system. In brief, electronic images were
digitized with a Leica CCD DC100 color video camera into a 1 kilopixel
1 kilopixel image buffer of the AlphaEase 5.02 image analysis
system (Alpha Innotech Corp). A color threshold mask for immunostaining was
defined to detect the red color by sampling, and the same threshold was applied
to all specimens. The percentage of the total area with positive color for each
section was recorded.
TABLE 1. Characteristics of Study Patients
Variable
Control (n
35)
Simvastatin (n
35)
Age, y
71
72
Male/female
18
- 17
19
-16
Patients with:
;
Recent TIA and stroke
35
35
Family history of IHD
18
16
Hypertension
25
23
Diabetes
12
13
Cigarette smoking
19
18
NSAID or glucocorticoid treatment 0
0
Stenosis severity, %
Mean +/-SD
76
+/-6
77
+/-8
Range
70–93
70–95
Percentage of macrophage-rich areas 26 +/-11*
8 +/-5
Number of T cells per mm2 section area 71 +/-21*
23
+/-11
Percentage of SMC-rich areas
19 +/-8+
15 +/-6
TIA indicates transient ischemic attack; IHD, ischemic heart
disease; and NSAID, nonsteroidal antiinflammatory drug. *P0.0001; †P0.03.
_____details left out, see journal at http://old.spread.it/Volume/chapt18/add_18/ref_18107.pdf
Results
Percentages of carotid diameter reduction did not differ between
the 2 groups (1.11.4% versus 0.81.1%). Baseline
lipid levels were similar in the 2 groups (Table 2). At the end of the study, total cholesterol
and LDL-C levels were significantly reduced in patients treated with
simvastatin (30% and 41%, respectively), whereas they did not change in
patients randomized to diet alone (Table 2). No patient in either group
developed any clinical events during the study.
. . . . . . . .
COX-2 Expression in Plaques Is
Reduced
by Simvastatin
After treatment, COX-2 was more abundant in control lesions
than in plaques from simvastatin-treated subjects (23.25.2% versus 6.12.2%, n
35; P0.0001; Figure 2). COX-2 accumulated in the activated
macrophages at the shoulder region in control subjects, whereas it was
localized primarily in SMCs in simvastatin-treated patients. Finally, Western
blot and reverse transcription–polymerase chain reaction analyses (Figure 3)
confirmed markedly lower COX-2 expression in simvastatin-treated plaques
(6158152 versus 1236321 densitometric units [DU] for protein expression, n
35; P0.0001). In contrast, no significant effects were
observed with respect to COX-1 expression (Figure 2).
mPGES Expression in Plaques Is
Reduced by Simvastatin
Immunohistochemistry revealed strong mPGES immunoreactivity
in all of the control plaques but only weak staining in the simvastatin-treated
plaques (19.63.2% versus 3.71.3%, n
35; P0.0001; Figure 2). In the control group, mPGES localized
in the plaque shoulder, an area characterized as macrophage rich. Only weak
mPGES expression was observed by Western blot in simvastatin-treated plaques
(Figure 3). In contrast, a 5-fold higher signal was demonstrated in control
plaques (946265 versus 5163194 DU, n
35; P0.0001).
Effect of Simvastatin on MMP
Expression in Plaques
MMP staining was significantly less abundant in lesions from
simvastatin-treated patients than in controls (Figure 2). Levels of MMP-2 and
MMP-9 in control plaques (24.64.7% and 26.25.6%, respectively; n
35) significantly exceeded (P0.0001) those in
simvastatin-treated plaques (6.41.9% and 7.22.5%, respectively; Table 1).
Immunoreactivity localized in the plaque shoulder, corresponding to areas of
intense macrophage infiltration. Effect of Simvastatin on MMP Activity in
Plaques The higher (P0.0001) MMP-2 and MMP-9 immunoreactivity documented by
Western blot in control plaques (6231284 versus 1894187 and 6745421 versus 2745
+/-347 DU, respectively; n
35; Figure 4A) does not necessarily correspond to enhanced
enzymatic activity, because all MMPs require activation before they can digest
their substrate. Thus, we used
zymography to demonstrate that control plaques contained activated MMPs (Figure
4B). In contrast, only weak positivity for activated MMPs was observed in
simvastatin-treated plaques (Figure 4B). Thus, the amount of inactive and active
MMP-2 (5247285 versus 1145 +/-275 and 2874 +/-302 versus 598 +/-124 DU,
respectively; n =35) and MMP-9 (6136
+/-368 versus 1321 +/-354 and 2645641 versus 876221 DU, respectively) was
significantly higher (P0.0001) in the control plaques.
Effect of Simvastatin on Plaque
Extracellular Components Sirius red polarization showed
increased collagen content in the sections of simvastatin-treated patients
compared with control patients (15.23.4% versus 8.13.8%, n
35; P0.0001). In contrast, the plaque content of lipid (Figure
5A) and oxLDL (Figure 5B) was significantly reduced after simvastatin therapy
(27.2 +/-11.5% versus 6.9 +/-4.1% and 25.3 +/-5.8% versus 10.7 +/-2.8%,
respectively; n
35; P<0.0001)
Discussion
We have previously reported that COX-2 and mPGES contribute
to the clinical instability of atherosclerotic plaques by promoting plaque
rupture induced by MMPs, key enzymes in the final step of this process. Now, in
the present report, we provide evidence for the critical involvement of
COX-2/mPGES in the process of plaque stabilization induced by simvastatin
therapy. The present findings are the first, to the best of our knowledge, to
(1) demonstrate by randomized study the anti-inflammatory effect of statins in
human carotid atherosclerotic plaques, (2) show a direct inhibitory effect of simvastatin
on COX-2/mPGES in human atherosclerotic lesion, and (3) relate the inhibition
of COX-2/mPGES to the reduction of MMP activity observed after statin therapy.
Concomitantly higher expression of COX-2/mPGES, MMP-2, and
MMP-9 was found in specimens obtained from the culprit carotid lesions of
patients randomized to diet alone compared with specimens obtained from
patients randomized to simvastatin. In particular, all plaques obtained from
simvastatin-treated patients exhibited only weak positivity, whereas only 3
(8.5%) of 35 plaques randomized to diet alone demonstrated intensity of enzyme
expression comparable to that observed in simvastatin-treated plaques. Notably,
the positive impact of statin-dependent MMP suppression was confirmed by the
parallel increment in plaque collagen content after simvastatin therapy. In the
present study, macrophages were more abundant in control plaques, always
outnumbered the lymphocytes, and
represented the major source of COX-2/mPGES and MMPs. Furthermore, the
site of inflammatory infiltration in control plaques was always characterized
by strong expression of HLA-DR antigens on inflammatory cells, which contrasted
with the low expression of HLA-DR elsewhere in the simvastatin-treated plaques.
These data suggest the ability of simvastatin to reduce the inflammatory
reaction in symptomatic plaques. In fact, in agreement with the difference in
COX-2/mPGES and MMP staining pattern, the histological milieu of the lesions
appears different with regard to cellularity, presence of foam cells,
cholesterol clefts, and collagen content but not in the degree of vessel
stenosis, which suggests that lesions treated with simvastatin or diet alone
are different only with regard to inflammatory burden and that differences in
plaque behavior stem from differences between simvastatin and diet alone in the
ability to influence the expression of 1 or more proteins capable of disrupting
plaque stability.
Previous studies have reported the ability of statins to
reduce atherosclerotic lesion evolution toward rupture. However, these studies did not provide any
evidence about the involvement of COX-2/mPGES in the pathophysiology of simvastatin-dependent
plaque stabilization. COX-2 is an intermediate enzyme in the metabolic pathway
of arachidonic acid, and the COX bioproduct PGH2 is further metabolized by other
isomerases to various prostanoids. Thus, the relative abundance of a specific
prostanoid rather than another is the result of the expression and activity of
its specific somerase, and the
concomitant expression of functionally coupled COX-2/mPGES is necessary for the
biosynthesis of PGE2 - dependent MMPs in the setting of atherosclerotic plaque.
Interestingly, because macrophages of the shoulder region contain most of the
COX-2/mPGES protein within the lesion, they emerge as the principal cellular
target of simvastatin in the context of plaque stabilization. This finding may
have functional importance and may contribute to explain the controversial experimental
and clinical findings associated with selective COX-2 inhibition, because
different cell types can regulate the production of different eicosanoids.
Endothelium predominantly releases PGI2 , an inhibitor of platelet activation
and cholesterol accumulation, and Belton et al reported that COX-2 is
responsible for the increase in PGI2 seen in patients with atherosclerosis. In
contrast, macrophages, not present in normal arterial tissue, produce an array of
prostanoids, including PGE2 , considered one of the most atherogenic
eicosanoids.
Prostanoids have potent actions on SMCs, regulating
contractility, cholesterol metabolism, and proliferation. Reduced expression of
COX might thus contribute to the decrease of lipid accumulation in lesional
SMCs (and macrophages), reducing formation of SMC- and macrophage-derived foam
cells within atheroma. On the other hand, antiproliferative and antimigratory actions
of COX products on SMCs suggest potential contributions of the inhibition of
this enzyme to the evolution of a lesion toward an SMC-enriched and
macrophage-depleted, and thus more stable, plaque. Furthermore, COX-2 can modulate angiogenesis
by synthesis of angiogenic factors and neovessel formation. consequently, COX-2 inhibition within the
lesion contributes to the reduction of new blood vessel formation, thus
inhibiting plaque expansion. More importantly, PGE2 , a predominant eicosanoid
of macrophages, induces in human atherosclerotic plaques the expression of
MMP-2 and MMP-9, enzymes considered crucial in the degradation of plaque
stability. Our description of a strong reduction of these MMPs in plaque treated
with simvastatin and found to be macrophagedepleted and COX-2/mPGES-negative
suggests that such arachidonate-dependent inhibition of MMPs by simvastatin may
operate in vivo. Interestingly, these data obtained in patients with only
modest increments in LDL-C are in accord with the results of the recent Heart
Protection Study and provide further support against the existence of an LDL-C threshold
below which reductions would not reduce risk. The present results are partially
in agreement with Crisby et al because they demonstrate reduced MMP-2 activity
in statin-treated plaques. However, in the present study, simvastatin also
significantly reduced MMP-9 activity and increased the plaque content of SMCs,
whereas in the study by Crisby et al, pravastatin failed to obtain these
results. Several hypotheses could be raised to explain these conflicting data. First,
the study by Crisby et al, despite being well designed, was a nonrandomized
study, and thus, hidden biases could explain the different results.
Alternatively, the different statin used, the stronger lipid-lowering effect,
or the longer period of treatment might
all contribute to explain the observed differences.
The present report is also in accord with recent in vitro
evidences demonstrating that atorvastatin may reduce inflammation by decreasing
COX-2 expression in SMCs, although it differs from the report of Degraeve et
al, which showed that mevastatin and lovastatin may upregulate COX-2 expression
in SMCs in vitro. These contradictory data suggest that different statins may
exert diverse effects on the complex signal transduction pathways involved in
COX-2 regulation.
In the present study, the hypothesis that plaque COX-2/
mPGES suppression by simvastatin was largely dependent on the reduction in
plaque cholesterol was supported by the in vitro experiments with mevalonate
and by the observation that reduction of COX-2/mPGES in plaque was associated
with comparable reduction in oxLDL content. However, further studies directly
comparing simvastatin with other lipid-lowering strategies are necessary to
definitively answer this question.
In conclusion, the present study addresses the missing link
between statin therapy and MMP inhibition that leads in turn to plaque
stabilization, by demonstrating the inhibition of thefunctionally coupled
COX-2/mPGES in human atherosclerotic lesions after simvastatin therapy and
providing evidence that downregulation of COX-2/mPGES in activated macrophages
by simvastatin is associated with plaque stabilization, possibly by suppression
of MMP-induced matrix degradation, which promotes plaque rupture. These
findings are potentially important from a fundamental standpoint because they
indicate a crucial role for inducible COX/PGES in the stabilization of
atherosclerotic lesions observed with statins. From a practical standpoint,
these findings provide further support for the possibility that statins might
provide a novelform of therapy for plaque stabilization in patients with
atherosclerotic disease.
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