Association of serum bilirubin with oxidant damage of human atherosclerotic plaques and the severity of atherosclerosis.

Bilirubin has protective effects against atherosclerotic cardiovascular diseases hypothetically due to its antioxidant-antilipoperoxidative properties. Thus, we investigated whether serum bilirubin is associated with oxidant damage, namely lipid peroxidation, of human atherosclerotic plaques and the severity of atherosclerosis. In this regard, we correlated the levels of serum total bilirubin (STB), direct (conjugated) bilirubin (SDB) and indirect (unconjugated) bilirubin (SIB) with those of fluorescent damage products of lipid peroxidation (FDPL) and lipid hydroperoxides (LOOH) of 32 endarterectomy-derived carotid atherosclerotic plaques. Moreover, we compared the levels of serum bilirubin and plaque lipoperoxides between two groups of patients of the study population with different severity of atherosclerosis as judged by the carotid stenosis degree, i.e., <90% (group A, n = 23) and ≥90% (group B, n = 9). Remarkably, the levels of STB were strongly inversely correlated with those of plaque FDPL (rS = -0.70, P < 0.0001) and LOOH (rS = -0.66, P < 0.0001), as were those of SIB (FDPL: rS = -0.68, P < 0.0001; LOOH: rS = -0.63, P < 0.0001). SDB had a weaker association with plaque FDPL (rS = -0.41, P < 0.05) and LOOH (rS = -0.35, P < 0.05). Moreover, the levels of STB, SDB and SIB were lower and those of plaque lipoperoxides higher in group B than in group A, pointing to the association of serum bilirubin and plaque oxidant burden with the severity of atherosclerosis. In conclusion, lowered serum bilirubin is associated with oxidant damage of human atherosclerotic plaques and the severity of atherosclerosis.

Reactive aldehyde-scavenging enzyme activities in atherosclerotic plaques of cigarette smokers and nonsmokers.

OBJECTIVE: To investigate enzymatic reactive aldehyde-scavenging enzyme capacity together with lipid peroxidation as expression of oxidative stress in atherosclerotic plaques of cigarette smokers and nonsmokers. METHODS: We have assessed specific enzymatic activities of class 1, 2, and 3 aldehyde dehydrogenase (ALDH1, ALDH2, and ALDH3, respectively), glutathione S-transferase (isozyme A4-4, GSTA4-4), and aldose reductase (AR), namely the major reactive aldehyde-scavenging enzymes, together with lipid peroxidation, i.e., fluorescent damage products of lipid peroxidation (FDPL), in carotid atherosclerotic plaques surgically removed from 17 cigarette smokers and 17 nonsmokers. RESULTS: The enzymatic activities of ALDH1 plus ALDH2, ALDH3, GSTA4-4, and AR were significantly lower in the atherosclerotic plaques of smokers than in those of nonsmokers, while plaque FDPL levels were significantly higher in the smokers than in the nonsmokers. The amount of cigarette smoking was correlated inversely with the aforementioned plaque enzymatic activities and directly with plaque FDPL content. Plaque FDPL levels were inversely correlated with plaque enzymatic activities in smokers and nonsmokers. The degree of carotid atherosclerotic stenosis, as expression of atherosclerosis severity, was correlated inversely with plaque enzymatic activities and directly with plaque FDPL levels in smokers and nonsmokers; moreover, the degree of carotid stenosis was directly correlated with the amount of cigarette smoking. CONCLUSION: atherosclerotic lesions of cigarette smokers are endowed with a depressed enzymatic reactive aldehyde-scavenging capacity eventually favoring oxidative stress and the severity of atherosclerosis.

Overexpression of microRNA-145 in atherosclerotic plaques from hypertensive patients.

BACKGROUND: MicroRNAs (miRNAs) are endogenous, non-coding, short, single-stranded RNAs and represent a new class of gene regulators. Recent evidence supports a role for miRNAs in cardiovascular pathophysiology and atherosclerosis development. We have previously demonstrated that miR-145 is widely expressed in human atherosclerotic lesions and its downregulation has been correlated with vascular smooth muscle cell dedifferentiation, a cardinal step in the development of atherosclerosis. However, no evidences are available at this time about modulation of miR-145 in the setting of hypertension. Thus, the aim of this study was to investigate the expression of miR-145 in complicated hypertension. MATERIALS AND METHODS: Atherosclerotic plaques were obtained from 22 patients undergoing carotid endarterectomy for high-grade internal carotid artery stenosis. Plaques were subdivided into hypertension (n = 15) and control (n = 7) groups according to the presence or absence of hypertension (as defined by blood pressure > 140/90 mmHg or current antihypertensive treatment). In study plaques, miR-145 values were evaluated using real-time PCR. The level of induction has been tested by using ΔΔ cycle threshold method. RESULTS: We found that miR-145 was significantly more expressed in atherosclerotic plaques of hypertensive patients than in control plaques (1.201 ± 0.260 vs 0.483 ± 0.148 fold induction ± SE; p = 0.026). Moreover, a post-hoc analysis showed that treatment with angiotensin receptor blockers may be associated with the maximum increase in miR-145 levels, although these data did not show any statistical significance probably due to the limited sample size. CONCLUSIONS: To the best of our knowledge, this study is the first demonstration that hypertension may upregulate miR-145 expression in human atherosclerotic plaques. Future investigations will be necessary to establish the molecular readout of miR-145 upregulation in atherosclerotic lesions in hypertension.

A unique microRNA signature associated with plaque instability in humans.

BACKGROUND AND PURPOSE: Atherosclerotic plaque rupture is considered the most important mechanism that underlies the onset of stroke, myocardial infarction, and sudden death. Several evidences demonstrated the pivotal role of inflammatory processes in plaque destabilization. MicroRNAs (miRNAs) are small endogenous RNAs and represent a new important class of gene regulators. Nevertheless, no data exist about the expression profile of miRNAs in atherosclerotic plaques. Thus, the aim of this study was to investigate the expression level of miRNAs in human plaques and to correlate it with clinical features of plaque destabilization. METHODS: Two separate groups of plaques were collected from patients who underwent carotid endarterectomy in Chieti (n=15) and Ancona (n=38) Hospitals. All the plaques were subdivided in symptomatic (n=22) and asymptomatic (n=31) according to the presence/absence of stroke. RESULTS: First, on the plaques collected at Chieti Hospital, we performed large-scale analysis of miRNA expression. Between the 41 miRNAs examined, we discovered profound differences in the expression of 5 miRNAs (miRNA-100, miRNA-127, miRNA-145, miRNA-133a, and miRNA-133b) in symptomatic versus asymptomatic plaques. Remarkably, when we repeated the analysis on the Ancona plaque subset, all these 5 miRNAs confirmed to be significantly more expressed in the symptomatic plaques. Finally, in vitro experiments on endothelial cells transfected with miRNA-145 and miRNA-133a confirmed the importance of these miRNAs in the modulation of stroke-related proteins. CONCLUSIONS: These results are the first to report alterations in the expression of specific miRNAs in human atherosclerotic plaques and suggest that miRNAs may have an important role in regulating the evolution of atherosclerotic plaque toward instability and rupture. Furthermore, by identifying the specific miRNA signature for stroke now, we are able to use computer algorithms to identify previously unrecognized molecular targets.

Serum albumin and biomolecular oxidative damage of human atherosclerotic plaques.

OBJECTIVES: To investigate the association of serum albumin (SA) with oxidative damage of human atherosclerotic plaques and the severity of atherosclerosis. DESIGN AND METHODS: Correlation of the levels of SA with those of lipid and protein oxidation of endarterectomy-removed carotid atherosclerotic plaques; SA and plaque oxidative biomarkers comparison between 2 groups of patients with different severity of atherosclerotic carotid stenosis, i.e. <90% (group I) or ≥90% (group II). RESULTS: SA was strongly inversely correlated with plaque oxidative damage; SA was lower and plaque oxidative damage higher in group II than group I. CONCLUSIONS: Lowered SA is associated with oxidative damage of atherosclerotic plaques and the severity of atherosclerosis.

Association of body iron stores with low molecular weight iron and oxidant damage of human atherosclerotic plaques.

The association between iron, an oxidant catalyst, and atherosclerosis is controversial. In particular, it is unknown whether: (1) stored iron, namely serum ferritin, is correlated with catalytic iron and oxidant damage of human atherosclerotic plaques; (2) catalytic iron is related to oxidative injury within such plaques; (3) plaque oxidant burden is associated with the severity of atherosclerosis. Thus, we assessed low molecular weight iron (LMWI), which represents the metal catalytically active form, together with fluorescent damage products of lipid peroxidation (FDPL) and lipid hydroperoxides (LOOH), in 38 atherosclerotic plaques surgically removed from 38 patients who had undergone selective carotid endarterectomy. In each patient, the levels of serum ferritin were measured and correlated with those of plaque LMWI and lipoperoxides by the Spearman rank correlation test with Spearman rank correlation coefficient (r(S)) calculation. Moreover, in patients selected from the same study population, we compared plaque analyte levels between two groups with different severity of atherosclerotic carotid stenosis, i.e., <90% (group A, n = 25) or > or =90% (group B, n = 13), and between another two groups without (group C, n = 27) and with (group D, n = 11) associated contralateral carotid stenosis > or =50%, indicative of "extensive" and more severe atherosclerotic disease. In group A patients, serum ferritin was directly and significantly correlated with plaque LMWI (r(S) = 0.46, P < 0.025) and FDPL (r(S) = 0.58, P < 0.005), while its correlation with plaque LOOH, albeit direct, did not attain statistical significance. Moreover, a direct and significant relationship was evident between the plaque content of LMWI and that of both FDPL (r(S) = 0.61, P < 0.0025) and LOOH (r(S) = 0.51, P < 0.025), suggesting a prooxidant role of catalytic iron within human atherosclerotic plaques. Considering the 13 patients of group B, a positive and significant correlation was observed between the levels of serum ferritin and those of plaque LMWI (r(S) = 0.83, P < 0.0001); on the other hand, serum ferritin, as well as plaque LMWI, showed no significant correlation with either plaque FDPL or LOOH, conceivably reflecting the small number of patients belonging to group B. Finally, plaque LMWI, FDPL, and LOOH content was significantly higher in group B than in group A, and in group D than in group C. These data suggest a role for catalytic iron in atherosclerotic plaque oxidation and in the severity of atherosclerosis, which appears indeed associated with plaque oxidant burden.

Suppression of RAGE as a basis of simvastatin-dependent plaque stabilization in type 2 diabetes.

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 with asymptomatic carotid artery stenosis (>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-kappaB, 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 PGE2-dependent MMPs, responsible for plaque rupture.

Association between prostaglandin E receptor subtype EP4 overexpression and unstable phenotype in atherosclerotic plaques in human.

OBJECTIVE: We recently demonstrated that inducible cyclooxygenase/PGE synthase-1 (COX-2/mPGES-1) are overexpressed in symptomatic plaques in association with PGE2-dependent metalloproteinase (matrix metalloproteinase [MMP]) biosynthesis and plaque rupture. However, it is not known which of the 4 PGE2 receptors (EP1-4) mediates macrophage metalloproteinase generation. The aim of this study was to characterize EP1-4 expression in plaques from symptomatic and asymptomatic patients undergoing carotid endarterectomy and correlate it with the extent of inflammatory infiltration, COX-2/mPGES-1 and MMP expression and clinical features of patients' presentation. METHODS AND RESULTS: Plaques were analyzed for COX-2, mPGES-1, EP1-4, MMP-2, and MMP-9 by immunohistochemistry, reverse-transcription polymerase chain reaction and Western blot; zymography was used to detect MMP activity. We observed strong EP4 immunoreactivity, only very weak staining for EP2, and no expression of EP1 and EP3 in atherosclerotic plaques. EP4 was more abundant in MMP-rich symptomatic lesions, whereas EP2 was no different between symptomatic and asymptomatic plaques. Finally, MMP induction by PGE2 in vitro was inhibited by the EP4 antagonist L-161 982, but not by its inactive analog L-161 983 or by the EP2 antagonist AH6809. CONCLUSIONS: This study shows that EP4 overexpression is associated with enhanced inflammatory reaction in atherosclerotic plaques. This effect might contribute to plaque destabilization by inducing culprit metalloproteinase expression.

Association between 5-lipoxygenase expression and plaque instability in humans.

OBJECTIVE: The participation of 5-lipoxygenase (5-LO) in the development of atherosclerosis has been suggested by recent studies. However, a role for 5-LO as a modulator of atherosclerotic plaque instability has not been previously reported in humans. Thus, the aims of this study was to analyze the expression of 5-LO in human carotid plaques and to investigate the mechanism by which this enzyme could lead to plaque instability and rupture. METHODS AND RESULTS: We obtained atherosclerotic plaques from 60 patients undergoing carotid endarterectomy. We divided the plaques into symptomatic and symptomatic according to clinical evidence of plaque instability. Clinical evidence of plaque instability was provided by the assessment of recent ischemic symptoms attributable to the stenosis and by the presence of ipsilateral cerebral lesion(s) determined by computed tomography. Plaques were analyzed for CD68+ macrophages, CD3+ T cells, alpha-actin+ smooth muscle cells, 5-LO, cyclooxygenase 2, matrix metalloproteinase (MMP)-2, and MMP-9 by immunohistochemical, immunoblotting, and densitometric analyses. MMP activity was assessed by zymography. Leukotriene (LT) B4 and collagen were quantified by ELISA and Sirius red polarization, respectively. The percentage of macrophage-rich and T-cell-rich areas was larger in symptomatic compared with asymptomatic patients (25+/-6% versus 8+/-4%, P<0.0001, and 74+/-17 versus 18+/-4 cell/mm2, P<0.003). 5-LO expression was higher in symptomatic compared with asymptomatic plaques (24+/-4% versus 6+/-3%, P<0.0001) and was associated with increased MMP-2 and MMP-9 expression (27+/-4% versus 7+/-3%, P<0.0001, and 29+/-5% versus 8+/-2%, P<0.0001) and activity and with decreased collagen content (6.9+/-2.4% versus 17.8+/-3.1%, P<0.01). Immunofluorescence showed that 5-LO and MMPs colocalize in activated macrophages. Notably, higher 5-LO in symptomatic plaques correlated with increased LTB4 production (18.15+/-3.56 versus 11.27+/-3.04 ng/g tissue, P<0.0001). CONCLUSIONS: The expression of 5-LO is elevated in symptomatic compared with asymptomatic plaques and is associated with acute ischemic syndromes, possibly through the generation of LTB4, subsequent MMP biosynthesis, and plaque rupture.

Increased expression of transforming growth factor-beta1 as a stabilizing factor in human atherosclerotic plaques.

BACKGROUND AND PURPOSE: Transforming growth factor-beta (TGF-beta) is a growth factor/cytokine involved in vascular remodeling and atherogenesis. Recent studies in apolipoprotein E-deficient mice have demonstrated a pivotal role of TGF-beta in the maintenance of the balance between inflammation and fibrosis in atherosclerotic plaques. Furthermore, inhibition of TGF-beta signaling has been shown to accelerate plaque formation and its progression toward an unstable phenotype in mice. However, if this mechanism is operative also in humans is still unknown. The aim of this study was to characterize the expression of TGF-beta1 in human carotid plaque and to correlate it with the extent of inflammatory infiltration and collagen content with the clinical signs of plaque instability. METHODS: Plaques were obtained from patients undergoing carotid endoarterectomy and divided into symptomatic and asymptomatic according to clinical evidence of recent transient ischemic attack or stroke. Plaques were analyzed for TGF-beta1 expression by Immunocytochemistry, Western, and Northern blotting analysis. Immunocytochemistry was used to identify CD68+ macrophages, CD3 T lymphocytes, HLA-DR+ cells, and alpha-smooth muscle cells. Procollagen and interstitial collagen content were analyzed by immunohistochemistry and Sirius Red staining, respectively. RESULTS: Plaque TGF-beta1 mRNA was increased up to 3-fold in asymptomatic as compared with symptomatic plaques. Plaques from asymptomatic group had fewer (P<0.0001) macrophages and T lymphocytes compared with symptomatic plaques. TGF-beta1 gene was transcriptionally active as demonstrated by increased (P<0.0001) TGF-beta1 protein expression in asymptomatic plaques. Immunohistochemistry showed that TGF-beta was mainly expressed in plaque shoulder and was associated with a comparable increase (P<0.0001) in plaque procollagen and collagen content. CONCLUSIONS: In conclusion, this study demonstrates the higher expression of TGF-beta1 in human asymptomatic lesions and provides evidence that TGF-beta1 may play an important role in the process of plaque stabilization.

A polymorphism in the cyclooxygenase 2 gene as an inherited protective factor against myocardial infarction and stroke.

CONTEXT: Myocardial infarction (MI) and ischemic stroke are thought to be caused by matrix digestion by metalloproteinases (MMPs) leading to rupture of atherosclerotic plaques. Production of macrophage MMP-2 and MMP-9 is induced by cyclooxygenase 2 (COX-2) and prostaglandin E(2) synthesis. Although COX-2 expression may be genetically determined, the relation between COX-2 polymorphisms and the risk of MI and stroke is unclear. OBJECTIVE: To investigate the relationship between the -765G-->C polymorphism of the COX-2 gene and clinically evident plaque rupture. DESIGN, SETTING, AND PARTICIPANTS: Prospective, matched case-control study conducted between March 2002 and October 2003 among 864 patients with first MI or atherothrombotic ischemic stroke and 864 hospitalized controls. The groups were matched for age, sex, body mass index, smoking, hypertension, hypercholesterolemia, and diabetes. The -765G-->C variant of the COX-2 gene was genotyped by restriction endonuclease digestion of polymerase chain reaction products. MAIN OUTCOME MEASURES: Presence of the -765G-->C polymorphism of the COX-2 gene; COX-2, MMP-2, and MMP-9 expression and activity in plaques and in peripheral monocytes; urinary 6-keto PGF1alpha (marker of endothelial prostacyclin); and endothelium-dependent and -independent forearm blood flow vasodilation. RESULTS: The prevalence of -765GC was 2.41 times higher among controls than among cases (43.3% vs 17.9%; P<.001). The prevalence of -765CC homozygosity was 5.81 times higher (6.4% vs 1.1%; P =.04). Among participants carrying the -765GC and -765CC genotypes, the prevalence ratios for MI or stroke were 0.48 (95% CI, 0.36-0.68) and 0.33 (95% CI, 0.24-0.55), respectively. Expression of COX-2 and MMPs was significantly lower in atherosclerotic plaques from participants carrying the -765C allele, while the -765G-->C polymorphism did not affect endothelial prostacyclin biosynthesis or endothelium-dependent vasodilation in vivo. In subgroup analyses (n = 224 cases), serum high-sensitivity C-reactive protein was significantly lower in patients carrying the -765C allele (mean [SD], 0.78 [0.1] vs 2.56 [0.4] mg/L; P =.04). CONCLUSIONS: We found that the -765G-->C polymorphism of the COX-2 gene is associated with a decreased risk of MI and stroke. Detection of this genotype may be useful for predicting genetic risk of MI and stroke.

Balance between PGD synthase and PGE synthase is a major determinant of atherosclerotic plaque instability in humans.

OBJECTIVE: Inducible cyclooxygenase (COX-2) catalyzes the first step in prostanoid biosynthesis and is considered a proinflammatory enzyme. COX-2 and type 1 inducible PGE synthase (mPGES-1) have a role in metalloproteinase (MMP) release leading to plaque rupture. In contrast, lipocalin-type PGD synthase (L-PGDS) has been shown to exert antiinflammatory actions. Thus, in this study we investigated whether a shift from a PGDS-oriented to a PGES-oriented profile in arachidonate metabolism leads to inflammatory activation in rupture-prone plaque macrophages. METHODS AND RESULTS: Atherosclerotic plaques were obtained from 60 patients who underwent carotid endarterectomy, symptomatic (n=30) and asymptomatic (n=30) according to evidence of recent transient ischemic attack or stroke. Plaques were analyzed for COX-2, mPGES-1, L-PGDS, PPARgamma, IkappaBalpha, NF-kappaB, and MMP-9 by immunocytochemistry, Western blot, reverse-transcriptase polymerase chain reaction, enzyme immunoassay, and zymography. Prostaglandin E2 (PGE2) pathway was significantly prevalent in symptomatic plaques, whereas PGD2 pathway was overexpressed in asymptomatic ones, associated with NF-kappaB inactivation and MMP-9 suppression. In vitro COX-2 inhibition in monocytes was associated with reduced MMP-9 release only when PGD2 pathway overcame PGE2 pathway. CONCLUSIONS: These results suggest that COX-2 may have proinflammatory and antiinflammatory properties as a function of expression of downstream PGH2 isomerases, and that the switch from L-PGDS to mPGES-1 in plaque macrophages is associated with cerebral ischemic syndromes, possibly through MMP-induced plaque rupture.

Blockade of the angiotensin II type 1 receptor stabilizes atherosclerotic plaques in humans by inhibiting prostaglandin E2-dependent matrix metalloproteinase activity.

BACKGROUND: Clinical trials have demonstrated that agents that inhibit the angiotensin II pathway confer benefit beyond the reduction of blood pressure alone. However, the molecular mechanism underlying this effect has yet to be investigated. Recently, we have demonstrated enhanced expression of inducible cyclooxygenase (COX) and prostaglandin (PG)E2-dependent synthase (COX-2/mPGES-1) in human symptomatic plaques and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. Thus, the aim of this study was to characterize the effect of the angiotensin II type 1 (AT1) receptor antagonist irbesartan on the inflammatory infiltration and expression of COX-2/mPGES-1 and MMPs in human carotid plaques. METHODS AND RESULTS: Seventy patients with symptomatic carotid artery stenosis were randomized to irbesartan (300 mg/d) or chlorthalidone (50 mg/d) for 4 months before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2, mPGES-1, MMP-2, and MMP-9, angiotensin II, AT(1), AT2, and collagen content by immunocytochemistry, Western blot, and reverse-transcriptase polymerase chain reaction, whereas zymography was used to detect MMP activity. Immunohistochemistry was also used to identify CD68+ macrophages, CD3+ T lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Plaques from the irbesartan group had fewer (P<0.0001) macrophages, T lymphocytes, and HLA-DR+ cells; less (P<0.0001) immunoreactivity for COX-2/mPGES-1 and MMPs; reduced (P<0.0001) gelatinolytic activity; and increased (P<0.0001) collagen content. It is worth noting that COX-2/mPGES-1 inhibition was observed after incubation in vitro with irbesartan but not with the selective AT2 blockade PD123,319. CONCLUSIONS: This study demonstrates that irbesartan decreases inflammation and inhibits COX-2/mPGES-1 expression in plaque macrophages, and this effect may in turn contribute to plaque stabilization by inhibition of MMP-induced plaque rupture.

Suppression of the functionally coupled cyclooxygenase-2/prostaglandin E synthase as a basis of simvastatin-dependent plaque stabilization in humans.

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 (P<0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells; less (P<0.0001) immunoreactivity for COX-2/mPGES and MMPs; reduced (P<0.0001) gelatinolytic activity; increased (P<0.0001) collagen content; and reduced (P<0.0001) lipid and oxLDL content. Interestingly, COX-2/mPGES inhibition by simvastatin was completely reversed by mevalonate in vitro. 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.