Published online before print June 15, 2006, doi: 10.1161/01.ATV.0000232501.08576.73
© 2006 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Dysregulation of the Ubiquitin-Proteasome System in Human Carotid Atherosclerosis
From the Divisions of Cardiovascular Diseases (D.V., J.H., M.G., D.M., K.S., L.O.L., A.L.), Neurosurgery (F.B.M.), and Nephrology and Hypertension (L.O.L.), Mayo Clinic College of Medicine, Rochester, Minn.
Correspondence to Amir Lerman, Division of Cardiovascular Diseases, Mayo Clinic Rochester, 200 First St SW, Rochester, MN 55905. E-mail lerman.amir{at}mayo.edu
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Abstract |
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Objective— The ubiquitin-proteasome system is the principal degradation route of intracellular and oxidized proteins, thus regulating many cellular processes conceivably important for atherosclerosis. The aim of this study was to evaluate the activity of ubiquitin-proteasome system in human carotid artery plaques in relation to oxidative stress and clinical manifestation.
Methods and Results— In carotid endarterectomy specimens from 83 asymptomatic and 94 symptomatic patients, content of ubiquitin, ubiquitin conjugates, matrix metalloproteases, and NADPH-oxidase-p67 was evaluated by immunoblotting; proteolytic proteasome activity by fluorometric assay; single and double immunostaining for ubiquitin conjugates, 3-nitrotyrosine, apoptosis, smooth muscle 
-actin, and macrophage CD-68, as well as Sirius Red staining for collagen were performed. Compared with asymptomatic patients, symptomatic patients showed a more unstable plaque phenotype, an increased degree of apoptosis, a significantly higher ubiquitin conjugates content (17.72±1.36 versus 10.99±1.04; P<0.001), and lower proteasome activity (5.01±0.70 versus 9.41±1.19 nmol AMC/mg protein/min; P<0.01). Ubiquitin conjugates content was directly correlated to NADPH-p67 and degree of apoptosis. Immunostaining revealed colocalization of ubiquitin conjugates and 3-nitrotyrosine, and accumulation of ubiquitin conjugates in smooth muscle cells and macrophages.
Conclusions— In human carotid plaques increased oxidative stress is associated with inhibition of the proteasome activity and accumulation of ubiquitin conjugates, particularly in symptomatic patients. These results suggest a possible role of the ubiquitin-proteasome system in influencing plaque stability.
We evaluated the ubiquitin-proteasome system in relation to atherosclerosis clinical manifestation. Carotid plaques from patients with symptomatic disease showed accumulation of ubiquitin conjugates and lower proteasome activity, along with increased oxidative stress and apoptosis, as compared with asymptomatic patients. These data suggest a potential interplay between oxidative stress and ubiquitin-proteasome system activity in atherosclerosis pathophysiology.
Key Words: atherosclerosis • carotid plaque • endarterectomy • oxidative stress • proteasome • stroke • ubiquitin
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Introduction |
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The ubiquitin-proteasome system (UPS) is responsible for the nonlysosomal degradation of the majority of intracellular proteins,1,2 thus playing a crucial role in the regulation of many cellular processes.3 The process of ubiquitination requires various enzymatic activities, involving specific proteins, ie, E1, E2, E3, which activate and transfer polyubiquitin chains to target proteins, leading eventually to the formation of a complex which is recognized and degraded by the 26S proteasome complex.4 This complex is composed of a 20S core particle that embodies the catalytic activity and 2 19S regulatory particles. The targets of the UPS include key regulators of cell cycle and apoptosis and various transcription factors, whose intracellular levels are finely tuned in the maintenance of the optimum equilibrium for cell division, growth, differentiation, signal transduction, and response to stress.3,5 Many of these processes are crucially involved in the onset, progression, and complication of atherosclerosis. In particular the UPS plays a key role in the activation of NF-
B,6 which has been associated with coronary7 and carotid8 plaque instability. Moreover, the UPS degrades many molecules and regulators of apoptosis and angiogenesis,3 crucial mechanisms of plaque formation and rupture.9,10 It is therefore conceivable that dysregulation of the UPS plays a role in atherosclerotic plaque progression and tendency to rupture. Indeed, increased expression of ubiquitin conjugates has been demonstrated in human coronary plaque responsible for lethal myocardial infarction as compared with noninfarct related coronary lesions.11
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A high rate of protein ubiquitination is associated with increased oxidative stress12 especially in neurological disorders13–15 and UPS has been demonstrated to be the principal system responsible for the degradation of oxidized proteins.16 However, high-level oxidative stress can also impair UPS by reducing proteasome activity.15,17 This could lead to intracellular accumulation of ubiquitinated substrates caused by an increased production and by a reduced degradation. As shown in the development of cataract,18 intracellular ubiquitinated damaged proteins may eventually accumulate, form aggregates rich in carbonyl residues and be further enriched with ubiquitin moieties. These insoluble complexes cross-link and precipitate in the cell, inducing cytotoxicity and causing disruption of cell functions in several diseases.19–21 Inhibition of proteasome activity is also associated with increased apoptosis22 in several cell types, including smooth muscle cells,23,24 a crucial mechanism in determining atherosclerotic plaque instability.25
It is an open question whether, in human atherosclerosis, an overactivation or an underactivation of the system is correlated with clinical manifestation of the disease. We previously demonstrated, in an autopsy-based study, an association between enhanced accumulation of ubiquitinated substrates and clinical manifestation of coronary artery disease.11 The study of autopsic specimens, however, allows just a semi-quantitative evaluation of tissue sections and does not allow the evaluation of oxidative stress parameters or proteasome enzymatic activity. Therefore, it is not clear, yet, whether the accumulation of ubiquitinated complexes in human plaques is related primarily to an increased ubiquitination of cell proteins or to a blockade of the proteasome degrading system.
The current study was designed to test the hypothesis that clinically unstable atherosclerotic plaques are characterized by an enhanced accumulation of ubiquitinated proteins and to evaluate if the presence of a reduced proteasomal proteolytic activity contributes to this accumulation. We therefore evaluated the relationship between the presence of ubiquitin conjugates in human carotid atherosclerotic plaques after endarterectomy and the activity of the 20S proteasome, the degree of endogenous oxidative stress, the phenotypic features of plaque instability and the clinical manifestation of the disease.
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Methods |
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For detailed methodology, please see http://atvb.ahajournals.org
Patients
The study was approved by the Mayo Foundation Institutional Review Board, and procedures followed institutional guidelines. Written informed consent was obtained before surgery.
We studied 177 carotid plaques specimens from patients undergoing carotid endarterectomy, following a previously described procedure.26 Patients were defined "symptomatic" in the presence of a cerebral ischemic event (stroke, transient ischemic attack [TIA] or amaurosis fugax) within 6 months before surgery, ipsilateral to the collected plaque, or "asymptomatic" in the absence of previous events.
Plaque Specimens
After surgery, plaques were halved at the site of the maximum plaque diameter. One half was fixed in formalin and embedded in paraffin for histology; the other half was immediately frozen at –80°C for subsequent tissue analysis. Plaque stability was evaluated by the tissue expression of matrix-metalloproteases (matrix metalloproteinase [MMP]-2 and MMP-9), macrophage infiltration, content of fibrous tissue, and degree of apoptosis.
Western Blotting
Equal amount of proteins from carotid lysates were resolved in a 10% SDS-Page gel and transferred to a nitrocellulose membrane. After autoclaving for 30' to increase the sensitivity of ubiquitin conjugates detection27 and blocking, membranes were probed to detect ubiquitin conjugates (polyclonal antibody, 1:1000; Sigma), ubiquitin (monoclonal antibody, 1:500; Covance), MMP-9 (polyclonal antibody, 1:5000, Chemicon), MMP-2 (monoclonal antibody, 1:7500; Chemicon), and NADPH-oxidase p67phox (polyclonal antibody, 1:500; Santa Cruz); protein loading control was evaluated using anti-ß-actin antibody (1:5000; Sigma). Anti-rabbit or anti-mouse (Amersham Life Sciences) antibodies conjugated to horseradish peroxidase were used as secondary antibodies, as appropriate. After developing with chemiluminescence and exposing to x-ray film, signals were analyzed using ImageJ software (National Institutes of Health). Figure 1 shows a representative western blotting in a subset of 9 patients. For quantification of large ubiquitin conjugates, the density of the membrane column was analyzed in each sample (Figure 1A) and normalized for one same sample repeated in every membrane, as control. On the basis of the signal obtained, we divided the whole population into quartiles of ubiquitin conjugates content. Immunoblotting results are expressed as ratios to actin signal.
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Proteasome Activity Assay
Chymotrypsin-like activity of the proteasome was assayed using a commonly available fluorometric kit (Chemicon), following company instructions.
Immunostaining
Carotid plaque sections were immunostained following standard procedures (http://atvb.ahajournals.org), to detect ubiquitin conjugates, 3-nitrotyrosine content, as well as plaque macrophages and smooth muscle cells. Normal rabbit or mouse immunoglobulin fractions were substituted to primary antibodies as negative control.
Primary antibodies used: anti-ubiquitin-(conjugates) (1:100; Sigma), anti-3-nitrotyrosine (1:250; Sigma), anti--SMA (1:500; Dako), and anti-CD68 (1:500; Dako).
Expression of Ubiquitin Conjugates in Smooth Muscle Cells and Macrophages
Representative slices of whole plaques and cell dispersions were treated for single and double immunofluorescence staining to detect ubiquitin-(conjugates) and their co-localization with -SMA and CD-68. Fluorescence was observed using a Zeiss LSM-510 confocal laser scanning microscope (Carl Zeiss, Inc, Oberkochen, Germany).
In Situ Detection of Apoptosis by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling Assay
Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL) method using a commercially available kit (Apoptag® Peroxidase In Situ Apoptosis Detection Kit; Chemicon) according to the vendor’s instructions.
Sirius Red Staining for Collagen Content
Sections were stained following standard Sirius red procedure (http://atvb.ahajournals.org). The content of collagen type I and III, identified by birefringence under polarized light,28 was evaluated as percent of the plaque area.
Statistics
For clinical data, Western blotting and proteasome activity variables were compared by use of the Student t test or the 
2 test. Comparison among multiple groups was performed by ANOVA followed by Tukey-Kramer post-hoc analysis. Correlation was calculated with Pearson product moment. Data are expressed as percentage or mean±SE for continuous variables and by percentage for qualitative variables. Statistical significance was assumed for P<0.05.
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Results |
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The study population included 83 asymptomatic and 94 symptomatic patients. Clinical characteristics and current medications of the population are shown in the Table. The symptomatic group had a significantly lower level of high-density lipoprotein cholesterol, a higher percentage of diabetes, and lower percentage of patients taking antioxidant vitamins. No differences were observed for the other clinical parameters and medication (Table). Thirty-four symptomatic patients experienced stroke, 46 TIA, and 18 amaurosis fugax.
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Markers of Plaque Stability
Atherosclerotic plaques from symptomatic patients were characterized by a more unstable phenotype (Figure 2). In particular, as compared with asymptomatic patients, they showed a significantly lower fibrous tissue content (9.8±1.5% versus 21.1±1.7% of plaque area; P<0.01; Figure 2A and 2B), higher apoptosis (20.5±2.2 versus 11.9±2.2% of nuclei; P<0.01; Figure 2C and 2D), compatible with previous findings,29 higher macrophage infiltration (8.8±1.2% versus 3.0±1.4% of area; P<0.01; Figure 2E and 2F), MMP-9 (2.01±0.41 versus 1.03±0.16; P<0.05), and, although not significantly, MMP-2 (1.37±0.25 versus 0.70±0.09; P=0.06) expression. Moreover, the percentage of plaque area positively stained for -SMA was significantly lower in symptomatic (7.7±1.1%) than asymptomatic patients (12.4±1.3%; P<0.05; Figure 2E and 2F). Apoptotic nuclei were localized in cells expressing CD-68 (macrophages) and -SMA (smooth muscle cells), as well as in cells negative for both markers.
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Ubiquitin-Proteasome System
Immunoblotting for Ubiquitin and Ubiquitin Conjugates
The expression of free (8.6 KDa) ubiquitin was similar between the study groups (Figure 1B and 3
A). On the contrary, there was a significantly higher content of ubiquitin conjugates in carotid plaques from symptomatic as compared with asymptomatic patients (17.72±1.36 versus 10.99±1.04, P<0.001, Figure 1A and 3B). As shown in Figure 3B, patients who experienced stroke (16.11±1.99), TIA (16.5±2.12), and amaurosis fugax (18.28±3.82) showed a significantly higher content of ubiquitin conjugates as compared with asymptomatic patients (P<0.05 versus asymptomatic for all groups).
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P<0.05 vs asymptomatic.Ubiquitin conjugates content was not significantly influenced by major stroke risk factors (age, sex, blood pressure, total, low-density lipoprotein and high-density lipoprotein cholesterol levels, smoke habit, diabetes mellitus) or principal cardiovascular drugs. Within the diabetic patients, a tendency to a reduced accumulation of ubiquitin conjugates was associated with the use of oral antidiabetic drugs (14.4±5.7) as compared with non treated patients (24.4±5.7), although not significant (P=0.09).
A direct correlation was observed between ubiquitin conjugates accumulation and percentage of cells undergoing apoptosis (r=0.56; P<0.01). Finally, a significant difference in the prevalence of past cerebrovascular clinical events was observed between the highest and the lowest quartile of ubiquitin conjugates expression, resulting in an odd ratio of 3.81 (95% CI, 1.36 to 10.88) for all events, 1.92 (0.62 to 6.66) for stroke, and 1.78 (95% CI, 0.69 to 4.82) for TIA.
Proteasome Activity Assay
Proteasome proteolytic activity was evaluated in 44 patients randomly selected among both asymptomatic (11 samples) and symptomatic patients (11 samples in each subgroup, ie, amaurosis fugax, TIA and stroke). Symptomatic patients showed a decreased proteasome proteolytic activity as compared with asymptomatic patients (5.01±0.70 versus 9.41±1.19 nmol AMC/mg protein/min, respectively; P<0.01; Figure 3C). Patients with a previous stroke showed the lowest proteasome activity (3.30±1.19 nmol AMC/mg protein/min; P<0.05 versus asymptomatic patients).
Immunohistochemistry for Ubiquitin Conjugates
Immunoperoxidase staining showed the presence of ubiquitin conjugates in human carotid plaques, with a clear prevalence in the regions surrounding the necrotic core, in the necrotic core and in the shoulder regions (Figure 4). Immunofluorescence demonstrated a co-localization of ubiquitinated proteins with both macrophage marker CD-68 (Figure 5A and 5B) and even to a greater level, with -SMA (Figure 5C and 5D), suggesting accumulation of ubiquitin conjugates in smooth muscle cells and macrophages.
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Oxidative Stress
Immunoblotting expression of p67, indicator of NADPH-oxidase content, was significantly increased in symptomatic patients (2.31±0.29) as compared with asymptomatic patients (1.13±0.17; P<0.01). Moreover, a significant direct correlation between p67 expression and ubiquitin conjugates content was observed (r=0.72, P<0.001). The presence of hypertension was also associated with an increased expression of p67 (hypertensives 2.08±0.18 versus normotensives 1.03±0.43; P<0.05) and a trend toward increased expression of p67 was observed in hypercholesterolemic (1.94±0.21) as compared with normocholesterolemic patients (1.85±0.31; P=0.07).
A partial co-expression of ubiquitinated proteins and 3-nitrotyrosine was detected, mainly in shoulder regions and necrotic core of the plaque (Figure 5E and 5F).
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Discussion |
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The current study shows that the content of ubiquitin conjugates in plaques from patients with symptomatic carotid atherosclerosis is significantly higher as compared with asymptomatic patients, and correlates with 3-nitrotyrosine and NADPH-oxidase p67 expression and with the percentage of cells undergoing apoptosis. Importantly, symptomatic patients, characterized by a more unstable plaque phenotype, showed an imbalance between the accumulation of ubiquitin conjugates and proteasome proteolytic activity, which was reduced. Although correlative, this study suggests a possible role of the endogenous UPS in human atherosclerosis, potentially participating in the regulation of plaque stability and clinical complication of the disease.
The balance of the UPS is dependent on the active process of proteins ubiquitination and their degradation by the proteasome system. An increased formation of ubiquitin conjugates has been demonstrated in the presence of increased oxidative stress12 especially in neurological disorders.14 Similarly, in the present study, we demonstrated increased accumulation of ubiquitin conjugates in human carotid plaques, associated with increased oxidative stress, underscored by the significant correlation between ubiquitin conjugates and NADPH-oxidase subunit p67 expression, one of the principal sources of superoxide anions in human atherosclerosis and by the partial co-expression of ubiquitinated proteins and 3-nitrotyrosine. Besides enhancing the oxidative damage of proteins and the subsequent ubiquitination, oxidative stress may lead to accumulation of ubiquitinated proteins via inhibition of proteasome enzymatic activity. Indeed, reduced 20S proteasome activity was observed in symptomatic patients, suggesting an imbalance between the load of ubiquitinated substrates and the ability of the proteasome to degrade them. It can be speculated that the oxidative protein damage is responsible for the activation of the UPS leading to accumulation of ubiquitinated proteins30 within the plaque. As long as the proteasome complex is able to efficiently clear the cells from ubiquitinated substrates, an equilibrium is maintained. In the presence of a higher level of oxidative stress, the proteasome degrading activity may become insufficient to clear the cell from damaged proteins. These in turn are progressively enriched with ubiquitin moieties, are further oxidized, and form hydrophobic and cross-linking aggregates. Experimental studies have demonstrated this process in human cells exposed to a proteasome inhibitor.31 Protein aggregates are difficult to unfold or degrade, precipitate in the hydrophobic fraction of the cell and eventually become virtually undegradable, leading to cytotoxicity.30,32 Several age-related degenerative diseases, such as Alzheimer disease and Parkinson disease,33–36 cataracts,37 and some cancers38 are characterized by protein aggregates formation and precipitation. In these degenerative processes the accumulation of protein deposits is frequently associated with oxidative modification and increased ubiquitination of proteins.38–40 Our study suggests that a similar process may play a role in human atherosclerotic plaques, which can be considered, along with inflammatory, a degenerative disease. Furthermore, in atherosclerosis these processes might also enhance the acute complications of the atherosclerotic plaque. Besides facilitating direct cytotoxicity through accumulation of protein aggregates, inhibition of the proteasome can increase cell apoptotic rate, thus further contributing to plaque instability.9 Accordingly, in the present study we found a direct correlation between the accumulation of ubiquitin conjugates and plaque apoptosis, suggesting a relation between the two phenomena.
Our study demonstrates an increased presence of ubiquitin conjugates both in smooth muscle cells and macrophages and an increased apoptosis, which was undergoing in both cell types; we can speculate that the resulting increased rate of cell loss might contribute to the weakening of the fibrous cap and the enlargement of the necrotic core, respectively.
This scenario suggests that the active and high rate of ubiquitination, in the presence of a functioning proteasome complex, seems to have a protective role against the complication of atherosclerosis, via the degradation of oxidized and damaged proteins. On the contrary, when the proteasome-dependent degradation of its substrates is impaired, the accumulation of oxidized and ubiquitinated protein aggregates may enhance the damage and foster plaque instability. So far, proteasome inhibitors have been used for the treatment of several human cancers;41 however, because of the short duration of the treatment in such conditions, it is impossible to evaluate their effect on cardiovascular events. Interestingly, in patients with HIV the use of protease inhibitors, which have been demonstrated to significantly inhibit the proteasome system,42 is associated with an increased cardiovascular risk independently from associated risk factors.43 It is intriguing to hypothesize that such an increased risk might be partly related to their anti-proteasome activity.
It is noteworthy that aging per se has been associated with an impairment of the proteasome activity in human fibroblasts44 and induction of proteasome expression reduces cell senescence.44 In the present study, however, we did not find any association between age and proteasome activity or ubiquitin conjugates expression. This is likely due to the relatively homogeneous elderly population of our study which does not allow detection of age-related differences.
The association found in the present work, between ubiquitin conjugates content and oxidative stress, partly through proteasome inhibition, suggests an additional mechanism through which oxidative stress could favor not only the development but also the complication of human atherosclerosis. An accumulation of damaged, oxidized and ubiquitinated proteins can lead to proteasome inhibition by inducing a clogging of the 20S proteasome core.45 Therefore, either directly or through the formation of big proteins aggregates, high levels of oxidative stress can eventually lead to a net reduction of the clearance of damaged proteins. Finally, because the inhibition of the proteasome activity can increase oxidative stress,46 a vicious cycle between decreased enzymatic activity of the proteasome and increased oxidative stress might onset.
Study Limitations and Clinical Implications
Considering the relative small population, it was not possible to consider all the possible confounding factors, including specific drugs, duration, and combination of treatments. However, the principal drug classes did not appear to influence per se the UPS. The associative nature of the present work does not allow drawing definitive conclusions on the role of a dysregulation of the UPS in the pathophysiology of atherosclerosis. However, considering the amount of literature demonstrating the importance of the UPS in regulating the principal processes of atherosclerosis and our results, although it cannot be completely ruled out a role of bystander, it is probable that the modulation of the system plays a major role in the disease and may become one of the future targets for the treatment of atherosclerosis. So far, UPS has been clinically studied mainly in cancer research and in a recent trial the proteasome inhibitor bortezomib was found effective in the treatment of relapsed multiple myeloma.47 However, UPS regulates also many processes that are fundamental in atherosclerosis development and complication. It has been hypothesized that the inhibition of the proteasome might have a favorable effect on atherosclerosis through, among other mechanisms, the inhibition of the NF-B system6 and the induction of apoptosis.22 This is likely in the early stages of atherosclerosis, when the inhibition of the inflammatory process and cell proliferation are crucial to reduce the onset of the disease. Moreover, an inhibitor of proteasome activity has been found to prevent neointimal proliferation and restenosis after arterial injury, highlighting the possible application of this drug class for the prevention of restenosis after angioplasty.23 However, an untargeted and nonspecific inhibition of the system does not seem to be desirable in the treatment of atherosclerosis. Therefore, it is crucial to develop UPS-modulating drugs, which are able to target the specific degradation of key regulators in the atherosclerotic process, in relation to the stage of the disease. In this view, similarly to cancer research,48 the goal of future cardiovascular pharmaceutical engineering should be the development of drugs interfering with the regulators of UPS substrate-specificity, particularly the different E3 ubiquitin conjugating enzymes or the ancillary proteins.
In conclusion, the current study highlights the association between oxidative stress and UPS imbalance in advanced stages of atherosclerosis. The correlated progressive accumulation of ubiquitinated proteins, which may eventually become cytotoxic, might play a role in the regulation of plaque stability and eventually clinical manifestation of the disease.
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Acknowledgments |
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The authors are grateful to Monica L. Olson and Timothy M. Borland for their technical help in collecting and processing endarterectomy specimens.
Sources of Funding
This work was supported by the National Institutes of Health (R01 HL 63911 and K-24 HL 69840-02), the University of Pisa (Italy), the Italian Society of Hypertension and the Mayo Foundation. Dr Amir Lerman is an Established Investigator of the American Heart Association.
Disclosures
None.
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Footnotes |
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Original received March 11, 2006; final version accepted June 2, 2006.
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