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J Am Coll Cardiol, 2006; 47:2444-2455, doi:10.1016/j.jacc.2006.01.073
(Published online 24 May 2006). © 2006 by the American College of Cardiology Foundation |
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* Department of Geriatrics and Metabolic Diseases, Section of Pathology, Second University of Naples, Naples, Italy
"Centro di Eccellenza Cardiovascolare,", Section of Pathology, Second University of Naples, Naples, Italy
Department of Experimental Medicine, Section of Pathology, Second University of Naples, Naples, Italy
Department of Biochemistry, Section of Pathology, Second University of Naples, Naples, Italy
|| Cardiovascular Surgery Unit, Sassari Hospital, Naples, Italy
¶ Cardiovascular Surgery Unit, Hospital V. Monaldi, Naples, Italy
# Department of Surgery, Second University of Naples, Naples, Italy
** Department of Biochemistry and Biophysics "F. Cedrangolo," Section of Anatomic Pathology, Second University of Naples, Naples, Italy
Manuscript received November 9, 2005; revised manuscript received January 25, 2006, accepted January 29, 2006.
* Reprint requests and correspondence: Dr. Raffaele Marfella, Via Emilio Scaglione 141, 80145 Napoli, Italy. (Email: raffaele.marfella{at}unina2.it).
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Abstract |
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BACKGROUND: The role of the ubiquitin-proteasome system, the major pathway for non-lysosomal intracellular protein degradation in eucaryotic cells, in the progression of atherosclerotic plaque to instability is unclear.
METHODS: Plaques were obtained from 40 symptomatic and 38 asymptomatic patients undergoing carotid endarterectomy. Symptomatic patients received 8 mg rosiglitazone (n = 20) or placebo (n = 20) for 4 months before scheduled endarterectomy. Plaques were analyzed for macrophages (CD68), T-lymphocytes (CD3), inflammatory cells (HLA-DR), ubiquitin-proteasome activity, nuclear factor kappa B (NFkB), inhibitory kappa B (IkB)-beta, nitrotyrosine, matrix metalloproteinase (MMP)-9, and collagen content (immunohistochemistry and enzyme-linked immunosorbent assay).
RESULTS: Compared with asymptomatic plaques, symptomatic plaques had more macrophages, T-lymphocytes, and HLA-DR+ cells (p < 0.001); more ubiquitin-proteasome activity and NFkB (p < 0.001); and more markers of oxidative stress (nitrotyrosine and O2– production) and MMP-9 (p < 0.01) along with a lesser collagen content and IkB-beta levels (p < 0.001). Compared with placebo-treated plaques, rosiglitazone-treated symptomatic plaques presented fewer inflammatory cells (p < 0.01); less ubiquitin, proteasome 20S, and NFkB (p < 0.01); less nitrotyrosine and O2– production (p < 0.01); and greater collagen content (p < 0.01), indicating a more stable plaque phenotype.
CONCLUSIONS: Ubiquitin-proteasome overactivity is associated with enhanced inflammatory reaction in symptomatic plaques. The inhibition of ubiquitin-proteasome activity in lesions of symptomatic patients by rosiglitazone is associated with plaque stabilization, possibly by down-regulating NFkB-mediated inflammatory pathways.
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Methods |
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70% luminal narrowing). The study group comprised 38 patients with asymptomatic carotid stenosis (asymptomatic group) and 40 patients who presented with symptoms of cerebral ischemic attack (symptomatic group, "symptomatic" according to North American Symptomatic Carotid Endarterectomy Trial [NASCET] classification). Endarterectomy was performed 120 to 140 days after the onset of symptoms in these patients. During the baseline interview, a previous stroke was assessed by asking, "Did you ever suffer from a stroke diagnosed by a physician?" Medical records of patients who answered yes were checked to verify the diagnosis. A history of transient ischemic attack (TIA) also was assessed during the baseline interview. All TIAs were reviewed by a neurologist. A neurologist reviewed information on all possible strokes. A stroke was diagnosed if the clinical symptoms and neuroimaging were positive. Asymptomatic patients underwent a baseline clinical examination as well as medical history and had never developed neurologic symptoms or cerebral lesions assessed by computed tomography. All patients, both symptomatic and asymptomatic, received computed tomography or magnetic resonance imaging. Percentages of carotid diameter reduction, procedural methods, concomitant therapy, and risk factors did not differ between the two groups (Table 1). By the time of surgery, all patients were taking long-term aspirin therapy (100 mg/day). The symptomatic patients were randomized to receive rosiglitazone (4 mg twice daily, n = 20) or placebo (n = 20) for 4 months according to human studies evaluating the carotid arterial intima-media thickness progression as well as circulating markers of inflammation after PPAR-gamma agonist treatments (12,15). After the treatment period, all patients had undergone endarterectomy. Fasting plasma glucose, insulin, and serum lipids, as well as insulin resistance index (16), were measured at baseline, monthly, and before endarterectomy. Written informed consent was obtained from all patients before each examination. The local ethics review committee approved the study.
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Immunohistochemistry. After the surgical procedure, samples were immediately frozen in isopentane and cooled in liquid nitrogen. Similar regions of the plaque were analyzed (Fig. 1). Serial sections were incubated with specific antibodies anti-ubiquitin, anti-proteasome 20S, anti-alpha smooth muscle actin (Sigma-Aldrich Corp., Milan, Italy), anti–HLA-DR, anti CD68 (Monoclonal Mouse Anti-Human CD68, clone PG-M1, Dako, Glostrup, Denmark), and anti-CD3 (Monoclonal Mouse Anti-Human CD3, clone PC3/188A, Dako); anti–IkB-beta; and anti-matrix metalloproteinase (MMP)-9 (Santa Cruz Biotechnology, Santa Cruz, California). Specific antibodies that selectively recognized the activated form of nuclear factor-kB (p65 and p50 subunits, Santa Cruz) were used. For each immunohistochemical experiment, a negative control was performed with the primary antibody omitted (data not shown). The specimens were analyzed by an expert pathologist (intraobserver variability 6%) blinded to the patient’s diagnosis.
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Biochemical assays. Plaques were lysed and centrifuged for 10 min at 10,000 g at 4°C. After centrifugation, 20 µg of each sample were loaded, electrophoresed in polyacrylamide gel, and electroblotted onto a nitrocellulose membrane. Each determination was repeated at least three times. Ubiquitin, IkB-beta, MMP-9, and nitrotyrosine levels were quantified in plaques using a specific ELISA kit (Santa Cruz; R&D Systems, Minneapolis, Minnesota; Imgenex, San Diego, California). Nuclear extracts from plaque specimens were obtained as described by Ohlsson et al. (17). We used a specific antibody that selectively recognizes the activated form of the NFkB subunit p65. In addition, we analyzed the expression of the activated p50 subunit by specific Trans-AM NF-kB p50 transcription factor assay kit (Active Motif, Rixensart, Belgium). For the quantitative measurement of the proteasome 20S activity, a specific sodium dodecyl sulfate activation kit (Boston Biochem, Cambridge, Massachusetts) was used. Nitrotyrosine was assayed into the plaque tissue with a kits supplied by Hycult Biotech (Uden, the Netherlands).
Macrophages extraction from atherosclerotic plaques. Macrophages were selectively extracted from plaques as described by de Vries et al. (18). Biochemical assay on cell homogenates for ubiquitin and proteasome 20S determinations were performed as illustrated earlier.
Measurement of O2–. Production of O2– was measured as the superoxide dismutase-inhibitable reduction of cytochrome c as previously described (19).
Isolation and culture of blood monocytes. Peripheral blood monocytes from 20 symptomatic and 20 asymptomatic patients were purified and cultured as described by Cipollone et al. (20). In brief: human monocytes were isolated from buffy coat preparations purchased from the National Blood Transfusion Service or from whole blood taken from laboratory donors. Mononuclear cells were first isolated by Ficoll-Hypaque sedimentation and then further purified by Percoll density fractionation. Monocytes were identified by flow cytometry analysis according to their characteristic forward and side scatter on a Becton Dickinson FACScan flow cytometer. Purity was further checked by expression of CD14 using monoclonal antibodies (Monoclonal Mouse Anti-Human CD14, Clone TÜK4, Dako). The purity of monocytes ranged between 80% and 88%. Monocytes from symptomatic patients (24 x 106/4 ml of DME) were cultured in the presence or absence of rosiglitazone (7.0 µM for 48 h) and in the presence or absence of MG132 (Calbiochem, 10 mmol/l stock solutions in dimethyl sulfoxide), a specific proteasome inhibitor (10 µM for 5 h). At the end of the incubation, adherent monocytes were scraped, collected, and lysed; and ubiquitin (Monoclonal anti-Ubiquitin, Clone 6C1, Sigma, ELISA), proteasome 20S, NFk-B (Anti-NfkBp65, Anti-NfkBp50. Santa Cruz), IkB-beta (Anti-IKKb, Sigma), and O2– production were evaluated.
Sirius red staining for collagen content. Sections were stained as described by Cipollone et al. (20). After dehydration, the sections were observed under polarized light after being placed on coverslips. The sections were photographed with identical exposure settings for each section.
Statistical analysis.
Data are presented as mean ± SD. Continuous variables were compared among the groups of patients with one-way analysis of variance for normally distributed data and the Kruskal-Wallis test for non-normally distributed data. The Kolmogorov-Smirnov test was used to assess whether continuous variables were normally distributed or not. We compared data using a Kruskal-Wallis test for triglycerides and insulin. When differences were found among the groups, Bonferroni correction was used to make pairwise comparisons. The Bonferroni correction was used in the comparison of ubiquitin, proteasome, p50, p65, IkB-beta, MMP-9, and O2– production values. A p value 
0.05 was considered statistically significant. All calculations were performed using the computer program SPSS 12 (SPPS Inc., Chicago, Illinois).
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Colocalization of proteasome 20S and ubiquitin with macrophages in symptomatic plaques. Serial sections of symptomatic plaques were incubated with the primary antibodies anti-ubiquitin, anti-proteasome 20S, and anti-CD68. The expression of both ubiquitin and proteasome 20S were associated with CD68+ macrophages in plaque sections (Fig. 4). Thus, these analyses confirmed the concomitant presence of ubiquitin and proteasome 20S in macrophages of symptomatic plaque.
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In vitro study. Higher levels of ubiquitin, p50, p65, and O2– production, as well as higher proteasome 20S and lower IkB-beta levels, were evidenced in peripheral blood monocytes from 20 symptomatic patients compared with monocytes from asymptomatic patients (p < 0.01). Levels of ubiquitin, p50, p65, and O2– production, as well as proteasome 20S activity, were significantly lower in monocytes from symptomatic patients incubated with rosiglitazone than in monocytes from symptomatic patients incubated without rosiglitazone (p < 0.01). The IkB-beta levels were significantly higher in the symptomatic group monocytes incubated with rosiglitazone than in symptomatic group monocytes incubated without rosiglitazone (p < 0.01) (Fig. 6). Levels of p50 and p65 levels were significantly lower in monocytes from symptomatic patients incubated with MG132 than in symptomatic patients’ monocytes incubated without MG132 (p50: 15.9 ± 1.4 ng/mg vs. 21 ± 1.7 ng/mg, p < 0.01; p65: 22 ± 2.9 ng/mg vs. 31.5 ± 1.6 ng/mg, p < 0.01), whereas IkB-beta (33.6 ± 7.9 ng/mg vs. 18 ± 2.2 ng/mg, p < 0.01) was significantly higher in the symptomatic group monocytes incubated with MG132 than in symptomatic group monocytes incubated without MG132. Although ubiquitin levels were higher in monocytes incubated with MG132, there were no statistically significant differences among the groups (429.9 ± 27.8 ng/mg vs. 414 ± 49.8 ng/mg, p = 0.2).
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Discussion |
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A previous postmortem study (11) has reported enhanced ubiquitin expression in unstable coronary plaques. However, the study did not provide any evidence about the specific pathway transducing environmental stimuli in ubiquitin-proteasome overexpression in subgroups of high-risk plaques such as those found in symptomatic patients. In our study, macrophages, T-lymphocytes, and HDLA-DR+ inflammatory cells were more abundant in symptomatic plaques and represented the major source of ubiquitin-proteasome activity, suggesting the presence of an active inflammatory reaction in symptomatic plaques. Concomitantly higher expression of ubiquitin and proteasome was found in human plaque macrophages obtained from the carotid lesions of patients with recent TIA or stroke compared with specimens obtained from asymptomatic patients. In agreement with the difference in ubiquitin-proteasome staining pattern, the histologic milieu of the lesions appears different with regard to cellularity, but not in the degree of vessel stenosis, suggesting that symptomatic and asymptomatic lesions are different only with regard to inflammatory burden. Hence, the differences in plaque behavior likely stem from differences in the presence of stimuli (i.e. oxidative stress, as evidenced by high O2– production and nitrotyrosine levels) for selective expression of ubiquitin-proteasome, capable of disrupting plaque stability via NFkB induction. Nuclear factor kappa B is normally bound to IkB in the cytosol; this binding prevents its movement into the nucleus (21). Various cellular stimuli, such as oxidative stress, induce ubiquitination of phosphorylated IkBs and subsequent degradation by the proteasome (22). Degradation of IkBs results in unmasking of the nuclear localization signal of NFkB dimers, which subsequently translocates to the nucleus, where it induces the transcription of proinflammatory cytokines that play a central role in plaque instability progression (23,24). Our findings also suggest that oxidative stress may induce phosphorylation and degradation of IkBs via the ubiquitin–proteasome overactivity, thus enhancing NFkB activation. According to the response-to-injury theory, atherosclerosis is initiated as an inflammatory-proliferative response to an injurious stimulus, constituted by cardiovascular risk factors such as hypercholesterolemia, hypertension, smoking, diabetes, and age (23). This injurious stimulus seems to be aggravation of endogenous oxidative stress, leading to the oxidative modification of lipids, proteins, and deoxyribonucleic acid, and thereby to structural and functional alterations within the vascular wall (25). Reduction in the bioavailability of nitric oxide (NO) due to increased production of reactive oxygen species is another important pathophysiologic element in atherogenesis (26). Under normal conditions, NO is constitutively produced by endothelial cells, antagonizing pro-atherosclerotic processes in part by suppressing the transcriptional activity of NFkB, which has been identified as a pivotal mediator in the inflammatory-proliferative process of atherogenesis (27). Reduction in NO bioavailability as well as alteration of cell signaling pathways due to an increase in oxidative stress, therefore, leads to the activation of NFkB (28). Previous reports evidenced the involvement of the ubiquitin–proteasome system in NFkB activation, particularly under conditions of aggravated oxidative stress. Oxidative stress is the common factor underlying hypercholesterolemia, hypertension, smoking, diabetes, and age and cardiovascular disease, and may explain the presence of inflammation in all these conditions (29). Although it is well recognized that inflammation is one manifestation of oxidative stress, and the pathways that generate the mediators of inflammation, such as interleukins, are all induced by oxidative stress (30), the mechanism by which oxidative stress may be involved in inflammatory process of symptomatic plaques is not fully clarified. In this context, our data suggest a novel mechanism by which oxidative stress, increasing ubiquitin-proteasome activity, may mediate inflammatory activity in symptomatic atherosclerotic plaques. Of note, it has been shown that oxidative stress can stimulate the ubiquitin system in macrophages by inducing the expression of components of its enzymatic machinery such as ubiquitin-binding proteins (31). Accordingly, in cultured monocytes from symptomatic patients we evidenced that O2– production as well as ubiquitin-proteasome activity and NFkB levels were significantly higher when compared to asymptomatic patients. Thus, we can speculate that increased ubiquitin-proteasome activity in plaque macrophage as a consequence of oxidative stress overexpression may enhance the synthesis of NFkB in the same cell, possibly representing a crucial step in the pathophysiology of plaque instability. Because of the study design, we cannot exclude whether the ubiquitin-proteasome pathway exerts also a protective compensatory response (32), or whether it is merely a correlative marker for the presence of inflammatory cells in symptomatic lesions. However, the concomitant presence of ubiquitin and proteasome 20S in macrophages of symptomatic patients, as well as the reduction of NFkB levels in monocytes from symptomatic patients treated with proteasome inhibitor suggests that the ubiquitin-proteasome pathway may have a pro-inflammatory effect in symptomatic lesions. Moreover, higher expression of ubiquitin-proteasome and MMP-9 in symptomatic plaques, one of the most important enzymes in the process of atherosclerotic plaque rupture (33), along with a lesser interstitial collagen content suggests an involvement of the ubiquitin proteasome system in instability of symptomatic lesion by increasing plaque erosion.
The present findings also show an inhibitory effect of rosiglitazone on ubiquitin-proteasome activity in symptomatic lesions. Indeed, symptomatic patients treated with rosiglitazone had the lowest level of ubiquitin and proteasome 20S activity, plaque inflammatory cells, cytokines, oxidative stress, and MMP-9 associated with the highest content of plaque interstitial collagen and VSMC content. Thus, patients assigned to rosiglitazone had less plaque progression to rupture compared with patients assigned to placebo. In particular, the reduced ubiquitin-proteasome activity seen in symptomatic plaques of the rosiglitazone group suggests decreased IkBs degradation and hence NFkB activation. All this may have clinical implications, because in a large series of carotid endarterectomy specimens, it has been shown that plaque inflammation is one of the major determinants of ischemic events in patients affected by carotid atherosclerotic disease (1). Sufficient data in humans suggest that PPAR-gamma agonists reduce common carotid arterial intima-media thickness progression as well as circulating markers of inflammation as early as three months after the administration in both nondiabetic and diabetic patients (12,15). Moreover, there are accumulating data to suggest that PPAR-gamma agonists exert anti-inflammatory and antioxidant effects, including decreases of cytokines and MMPs in monocytes, macrophages, T-lymphocytes, and VSMC (34,35). Although PPAR-gamma agonists may inhibit proteasome activity in human cancer (36), until now there was no evidence that this effect was reproducible in human atherosclerotic plaques. The hypothesis that plaque ubiquitin-proteasome activity is reduced by PPAR-gamma agonists is also supported by our in vitro experiment that evidenced a reduction of ubiquitin-proteasome activity, NFkB levels, and O2– production associated with a significant increment of IkB-beta in monocytes from symptomatic patients treated with rosiglitazone. The mechanism of repression of the proteasome by rosiglitazone remains a mystery and requires further studies. On the other hand, the reduction in O2– production by monocytes after rosiglitazone treatment may allow a lower polyubiquitination, as evidenced by reduction of ubiquitin levels. We therefore speculated that proteasome reduction by rosiglitazone may be induced by inhibition of oxidative stress and polyubiquitination. Rosiglitazone treatment also produced a significant reduction in insulin resistance, as indicated by reduced insulin resistance index levels. As insulin resistance is associated with increased cardiovascular and cerebrovascular risk (37), improved insulin sensitivity may be one mechanism by which rosiglitazone retards atherogenesis progression. On the other hand, oxidative stress has been shown to induce insulin resistance through NFkB activation (38): in this perspective, rosiglitazone, by reducing oxidative stress and ubiquitin-proteasome activity, may enhance both insulin sensitivity and symptomatic plaque stability. We also do not exclude the possibility that rosiglitazone inhibits NFkB activation through additional mechanisms independent of PPAR-gamma. Indeed, a PPAR-gamma–independent pathway may be operative in neuronal cells via inhibition of inducible nitric oxide synthase (39). Furthermore, PPAR-gamma agonists inhibit the nuclear translocation and subsequent deoxyribonucleic acid binding of NFkB via an IkBs-dependent pathway by inhibiting the immune response-induced degradation of IkBs (40). This provides evidence that a component of the anti-inflammatory effect is mediated by the action of PPAR-gamma agonists on the systemic immune system. Although further investigation will be required to elucidate the upstream mechanisms by which PPAR-gamma inhibits the degradation of IkBs, inhibition of cytokine gene expression suggests that activation of PPAR-gamma may alter functional elements common to both these pathways.
This study demonstrates enhanced ubiquitin-proteasome activity in symptomatic atherosclerotic lesions and provides evidence that the activation of this system by macrophages is associated with an NFkB-dependent increase in inflammation, potentially promoting plaque rupture. Moreover, the present study addresses the missing link between thiazolidinedione therapy and NFkB activity, which leads in turn to plaque stabilization, by demonstrating the inhibition of the ubiquitin-proteasome activity in human atherosclerotic lesions of symptomatic patients after rosiglitazone therapy and providing evidence that down-regulation of ubiquitin-proteasome activity is associated with plaque stabilization, possibly by suppression of NFkB-induced inflammation. These findings are potentially important from a fundamental standpoint because they indicate a crucial role for the inhibition of ubiquitin-proteasome activity in the stabilization of atherosclerotic lesions observed with thiazolidinediones. From a practical standpoint, these findings provide further support for the possibility that thiazolidinediones might provide a novel form of therapy for plaque stabilization in patients with atherosclerotic disease. However, these results require further confirmation from studies evaluating the effects of thiazolidinediones on a higher number of patients.
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12,14-prostaglandin J2. Proc Natl Acad Sci USA 1999;96:4668-4673.This article has been cited by other articles:
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  C. Di Filippo, R. Marfella, and M. D'Amico Possible Dual Role of Ubiquitin-Proteasome System in the Atherosclerotic Plaque Progression J. Am. Coll. Cardiol., October 14, 2008; 52(16): 1350 - 1351. [Full Text] [PDF]
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 J. E. Kanter, M. M. Averill, R. C. LeBoeuf, and K. E. Bornfeldt Diabetes-Accelerated Atherosclerosis and Inflammation Circ. Res., October 10, 2008; 103(8): e116 - e117. [Full Text] [PDF]
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J. Herrmann, S. M. Soares, L. O. Lerman, and A. Lerman Potential Role of the Ubiquitin-Proteasome System in Atherosclerosis: Aspects of a Protein Quality Disease J. Am. Coll. Cardiol., May 27, 2008; 51(21): 2003 - 2010. [Abstract] [Full Text] [PDF]
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 R. Marfella, C. Di Filippo, M. Portoghese, F. Ferraraccio, B. Crescenzi, M. Siniscalchi, M. Barbieri, C. Bologna, M. R. Rizzo, F. Rossi, et al. Proteasome Activity as a Target of Hormone Replacement Therapy-Dependent Plaque Stabilization in Postmenopausal Women Hypertension, April 1, 2008; 51(4): 1135 - 1141. [Abstract] [Full Text] [PDF]
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