- Email: [email protected]
Oxidized LDL in human carotid plaques is related to symptomatic carotid disease and lesion instability
Oxidized LDL in human carotid plaques is related to symptomatic carotid disease and lesion instability Fragiska Sigala, MD, PhD,a Athanassios Kotsinas, BSc, PhD,b Paraskevi Savari, MD,b Konstantinos Filis, MD, PhD,a Sophia Markantonis, BSc, PhD,c Efstathios K. Iliodromitis, MD, PhD,e Vassilis G. Gorgoulis, MD, PhD,b and Ioanna Andreadou, BSc, PhD,d Athens, Greece Background: Oxidative stress is an important determinant in atherosclerosis development. Various markers of oxidative stress, such as oxidation of low-density lipoprotein (LDL), nitrosative stress, lipid peroxidation, and protein oxidation, have been implicated in the initiation and/or progression of atherosclerosis, but their association with plaque erosion and symptomatic carotid disease has not been fully defined. In addition, certain oxidative markers have been shown in various models to promote plaque remodeling through matrix metalloproteinase (MMP) activation. Objective: To perform a global investigation of various oxidative stress markers and assess for potential relationships with destabilization and symptomatic development in human carotid plaques. Methods: Thirty-six patients undergoing endarterectomy were evaluated and compared with 20 control specimens obtained at the time of autopsy. Differences between stable and unstable plaques, symptomatic and asymptomatic patients, and >90% and <90% stenosis were evaluated. Oxidized LDL (ox-LDL), nitrotyrosine (NT), malondialdehyde (MDA), and protein carbonyls (PCs) levels were determined in atheromatic plaques homogenates by corresponding biochemical assays. Immunohistochemical (IHC) analysis was also employed to determine the percentage and topological distribution of cells expressing NT and metalloproteinase-9 (MMP-9) in serial sections from corresponding atheromatic plaques. MMP-9 expression was further verified using Western blot analysis. Results: Ox-LDL was increased in symptomatic patients (P < .05). Also, ox-LDL and NT levels were significantly higher in unstable versus stable carotid plaques (P < .05, respectively). Furthermore, IHC serial section analysis, corroborated by statistical analysis, showed a topological and expressional correlation between NT and MMP-9 (P < .05). MDA and PCs levels, although increased in carotid plaques, did not distinguish stable from unstable carotid plaques as well as symptomatic from asymptomatic patients with various degrees of stenosis. Conclusion: All types of investigated oxidative stress markers were significantly increased in human carotid plaques, but only ox-LDL levels were associated with clinical symptoms, while peroxynitrite products and MMP-9 were specifically related to plaque instability. ( J Vasc Surg 2010;52:704-13.) Clinical Relevance: Our results suggest that specific oxidative factors should be taken into consideration as additional future potential markers that could be used to assist in prevention and therapeutic decision.
Cardiovascular mortality is the major cause of death in adults older than 65 years in Western societies.1 AtheroscleFrom the First Department of Propaedeutic Surgery, Hippocrateion Hospital, Medical School, University of Athens,a the Molecular Carcinogenesis Group, Department of Histology-Embryology, Medical School, University of Athens,b the Laboratory of Biopharmaceutics and Pharmacokineticsc and the Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens,d the Second University Department of Cardiology, Medical School, University of Athens, Attikon Hospital.e Supported by National Kapodistrian University of Athens-Special Account for Research Grants (NKUA-SARG) grants No 70/4/9926, 70/4/ 9900, 70/4/9913, and 70/4/4281. VGG is also supported by the European Commission Grant FP7 INFLA-CARE (NKUA-SARG code 70/3/9815). Competition of interest: none. Reprint requests: Vassilis G. Gorgoulis, Antaiou 53 Str., Lamprini, Ano Patissa, GR-11146 Athens, Greece 15771 (e-mail: [email protected]) and Ioanna Andreadou, Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimioupolis, Zografou, Athens, Greece (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a competition of interest. 0741-5214/$36.00 Copyright © 2010 by the Society for Vascular Surgery. doi:10.1016/j.jvs.2010.03.047
704
rotic disease of the carotid artery is responsible for 20% to 30% of all strokes.2 At present, the only established criterion for predicting stroke is the degree of carotid stenosis.3 It seems, however, that plaque instability is an essential determinant of clinical manifestation of symptomatic carotid occlusive disease.4 The remodeling of a stable asymptomatic lesion into an unstable, ruptured plaque is a complex process.5,6 A plethora of data5,7,8 have implicated a variety of factors in plaque destabilization and eventually plaque rupture. Increasing evidence has shown that inflammation has a crucial role in the cascade of events related to plaque erosion.5,6 However, the molecular mechanisms that induce inflammation and subsequent plaque instability have not yet been completely deciphered. Available data have pointed to, among other mechanisms, the oxidative stress in these processes.9 Oxidation of low-density lipoprotein (LDL) has been shown to be a key factor for the atherogenetic development.10,11 Various reactive intermediates, derivatives of reactive oxygen (ROS) and/or reactive nitrogen (RNS) species, are known to mediate the oxidative modification of LDL.6,12,13 Among
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
Sigala et al 705
Table. Patients’ demographics and clinicopathological data Patients Demographic data Number Mean age (range) Male/female Smoking Current/past smokers Clinical data Hypertension Diabetes Hyperlipidemia Ischemic heart disease Aneurysm Peripheral arterial occlusive disease Clinical symptoms Stroke Transient ischemic attack Amaurosis fugax Angiographic carotic stenosis ⬍90% ⱖ90% Computed tomography brain (positive/negative) Plaque histopathology status Unstable Stable Medications Statins Angiotensin-converting enzyme inhibitors -blockers
Overall
Symptomatic
Asymptomatic
36 67.05 (55-80) 31/5 28 12/16
19 67.2 16/3 16 9/7
17 66.9 15/2 12 5/7
31 10 18 20 3 11 19 6 9 4
15 5 10 10 2 7 19 6 9 4
16 5 8 10 1 4 — — — —
16 20 19/17
7 11 19/0
17 19
14 3
3 16
18 15 19
10 7 10
8 8 9
them, peroxynitrite, the reaction product between nitric oxide (NO) and superoxide anion radicals,14 and malondialdehyde, a product from lipid peroxidation,10 are powerful LDL oxidants, which generate modified LDL with increased atherogenic potential.10,11 Interestingly, oxidizedLDL (ox-LDL) can lead to further production of ROS, thus creating a sustained oxidative stress condition.6 Of note, activation of matrix metalloproteinases (MMPs) has been shown to contribute also to plaque destabilization, through extracellular matrix remodelling.5,7 In certain cardiovascular experimental models, this activation was nitrosative stress-intermediates dependent,15-17 while in others, ox-LDL has been shown to be responsible for their expression.18,19 Other forms of oxidative stress, such as lipid peroxidation and protein oxidation, have been described in atherogenesis,10,20 but their contribution in this process, and specifically in symptomatic carotid disease and plaque instability generation, requires further investigation. To the best of our knowledge, a global assessment of various oxidative stress markers directly in human carotid plaques stratified according to their histologic status, degree of stenosis, and patients’ clinical status, is missing. Therefore, in the present study, we first sought to collectively determine the levels of various biochemical markers related to oxidative stress in the same clinical setting of human carotid plaques. These markers included ox-LDL, along with nitrotyrosine (NT) - the fingerprint of peroxynitrite, malondialdehyde (MDA), and protein carbonyls
9 0 0/17
(PCs), as indicators of nitrosative stress, lipid peroxidation, and protein oxidation, respectively. Subsequently, we looked into possible association of these markers with expression of MMP-9 and plaque destabilization in human carotid artery disease. MATERIALS AND METHODS Tissue collection Patients. Between 2007 and 2009, carotid plaques were prospectively collected from 36 random patients, who had internal carotid artery stenosis ⬎70% and underwent carotid endarterectomy. Demographic data, clinicopathological data, medication, risk factors, and vascular comorbidities were recorded (Table). All patients were evaluated preoperatively by a neurologist to be assigned as symptomatic and asymptomatic. Symptomatic patients were classified based on the presence of stroke, transient ischemic attacks, and amaurosis fugax. They also underwent a cerebral CT scan for identification of brain infarcts. Arteriography of the carotid bifurcation was performed in all patients for this study. Degree of stenosis was determined according to North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria.3 Based on these measurements, stenotic lesions were divided into two subgroups (70%-90% and ⱖ90%-99%), as previously described.21 The study protocol was approved by the Institutional Ethics Committee and all patients enrolled gave their informed consent.
JOURNAL OF VASCULAR SURGERY September 2010
706 Sigala et al
Control tissues. Twenty healthy carotid arteries obtained from autopsies served as control tissue samples for the “baseline-expression” of the examined factors in human arteries. Criteria for their selection included no previous history of cardiovascular disease and cause of death unrelated to pathologic conditions (eg, accidents), and were matched by gender (18 males and 2 women) and age (mean, 65.8 years; range, 50-74 years) to the patients’ specimens under investigation. Tissue preparation All carotid plaque specimens were removed in the operating room and were divided into two portions. One portion was fixed immediately in 10% neutral-buffered solution with 4% formaldehyde for 24 hours, and embedded in paraffin. The second portion was immediately stored at ⫺70oC for further analysis of ox-LDL, MDA, NT, and PCs and Western-blot analysis. Histology Hematoxylin and eosin staining was performed for histological evaluation of the specimens. Two pathologists, blinded to the clinical data, examined each specimen to assess atheromatous plaque morphology, using the American Heart Association classification of atherosclerotic plaques.22 According to this classification, carotid plaques were assigned as fibroatherotic (type V) and complicated (type VI). The latter type included plaques with intraplaque hemorrhage, ulcer, or thrombus, which were considered unstable. Immunohistochemistry Antibodies. For immunohistochemical analysis the following antibodies were used: anti-Nitrotyrosine (HM11; Zymed, Anti-Sel, Athens, Greece) at a 1:50 dilution, MMP-9 sheep polyclonal antibody (SantaCruz Biogenesis, Greece), in a 1:100 dilution. In serial sections, we also determined the cellular phenotype of atherosclerotic plaques. To identify macrophages/foam cells, smooth muscle cells, and endothelial cells, the following mouse monoclonal antibodies were used at a 1:50 dilution, respectively: CD68 (KP1; Dako, Kalifronas, Greece), alphasmooth muscle actin (1A4; Dako), and CD34 mouse (QBE nd/10; Biogenex, Aenorasis, Athens, Greece).7,23 Method. Immunohistochemistry was performed according to the indirect streptavidin-biotin-peroxidase method. In brief, 5 m paraffin sections were placed on poly-L-lysine-coated slides, dewaxed, rehydrated, and incubated for 30 minutes with hydrogen peroxide 0.3% to quench the endogenous peroxidase activity. Unmasking of the related proteins was carried out. The sections were incubated with the primary antibody at 4°C overnight. Biotin-conjugated secondary antibody was added at 1:200 dilution for 1 hour at room temperature (RT). The next stage comprised 30 minutes incubation in StreptAB Complex (1:100 stock biotin solution, 1:100 stock streptavidinhyperoxidase solution; Dako). For color development, we used 3,3=-diaminobenzidine tetrahydrochloride (DAB,
Sigma-Hellas, Athens, Greece) and hematoxylin as a counterstain. Evaluation. The staining pattern was considered positive only if cytoplasmic signal was discerned. Images were obtained with a Zeiss-Axiolab microscope (Carl Zeiss GmbH, Jena, Germany), employing a video analysis software as previously described.8 The NT and MMP-9 labeling indices (LI) were determined as the percentage of cells with cytoplasmic immunopositivity, irrespective of staining intensity. Of note, MMP-9 extracellular matrix staining was also observed and considered specific, but was not estimated in terms of a LI. Two independent observers (AK and VG) carried out slide examination. Inter-observer variability was minimal. Immunoblotting Protein extraction. Total protein extraction was performed according to the protocol described elsewhere.7 Briefly, tissues were homogenized in 10 mM TrisHCl pH 8, 170 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 0.5% nonidet P-40, 1 mM dithiothreitol and centrifuged at 1000 ⫻ g at 4°C for 5 minutes. The supernatant was collected and adjusted to 1g/mL protease and phosphatase inhibitors (Sigma-Hellas). Total protein concentration was calculated using the Roti-Quant Bradford-Assay (Roth, BioSure, Athens, Greece). SDS-PAGE. Thirty g of protein from total extracts of each sample were adjusted with NuPAGE LDS Sample Buffer (Invitrogen, Anti-Sel, Athens, Greece) and loaded on 4% to 12% gradient NuPAGE precast gels (Invitrogen). Gel electrophoresis and transfer to polyvinylidene difluoride membrane (Millipore, AlterChem, Athens, Greece) were performed according to standard protocols. Blots were blocked overnight in 2.5% gelatin solution in Trisbuffered saline (Sigma-Hellas) at room temperature. Subsequently, membranes were incubated for 3 hours with primary antibody solution in Tris-buffered saline supplemented with 0.5% Tween-20 at RT, followed by a 45 minute incubation with HRP-conjugated anti-mouse secondary antibody (1:2500 dilution; R&D Systems, Anti-Sel, Athens, Greece) at RT. Signal development was performed with the Western Glo-Chemiluminescent Detection Substrate (R&D Systems).7,23 Antibodies. The antibodies used were: anti-MMP-9 (1:500; sheep polyclonal, Neomarkers, BioAnalytica, Athens, Greece) and anti-tubulin (1:1000; AC-15, Abcam, Anti-Sel, Athens, Greece) for assessing equal loading of total protein and for normalizing the MMP-9 levels. Ox-LDL determination Ox-LDL was determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturers’ instructions (Biomedica, Biomedica Gruppe, Vienna, Austria). The detection limit of the assay was 0.8 ng/mL. NT determination NT, the fingerprint of peroxynitrite in the carotid plaques, was determined by ELISA according to the man-
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
ufacturers’ instructions (Bioxytech, Nitrotyrosine-EIA; Oxis Research, Beverly Hills, Calif). The detection limit of the assay was 2 nM. Measurement of carotid MDA Human carotid samples were frozen at ⫺70oC until the assay. On analysis, tissue samples were washed in ice-cold NaCl 0.9%, blotted on absorbent paper, and weighed. Each sample was minced in ice-cold 20 mM Tris-HCl buffer pH 7.4, in a 1:10 weight/volume ratio, and homogenized using a Teflon pestle. After centrifugation at 3000 ⫻ g for 10 minutes at 4oC, the supernatant was used for the biochemical assay.24 MDA concentration was determined spectrophotometrically at 586 nm and expressed in M (Oxford Biomedical Research Colorimetric Assay for lipid peroxidation) with some modifications.23 The method used was based on the reaction of the chromogenic reagent N-methyl2-phenylindole with MDA. One molecule of free MDA combines with two molecules of the chromogenic agent to produce a stable chromophore with a maximal absorbance at 586 nm. Briefly, 0.65 mL of 10.3 mM N-methyl-2-phenylindole in acetonitrile was added to 0.2 mL of tissue homogenate. After vortexing for 3 to 4 seconds and adding 0.15 mL of HCl 37%, samples were mixed well, closed with a tight stopper, and incubated at 45o C for 60 minutes. The samples were then cooled on ice, centrifuged, and the absorbance was measured spectrophotometrically at 586 nm. A calibration curve, comprising accurately prepared standard MDA solutions (from 2 to 20 nmol/mL), was also run for quantitation. Measurements of each group were performed in triplicate. Determination of protein carbonyls content A small amount of tissue was rinsed with phosphatebuffer saline solution to remove red blood cells or clots. Tissue was homogenized in 1 mL of cold phosphate-buffer pH 6.7, containing 1 mM EDTA, and then centrifuged at 10,000 ⫻ g for 15 minutes at 4oC. The supernatant was removed, and the tissue was stored at ⫺80oC until use. A modification of the technique of Levine et al,25 based on spectrophotometric measurement of 2,4-dinitrophenylhydrazine (DNPH) derivatives of protein carbonyls, was used to quantify proteincarbonyl content. Briefly, 100 L of plasma was incubated either with 500 L DNPH or 2 N HCl (blank) for 1 hour at RT. The samples were then reprecipated with 600 l 20% trichloroacetic acid (TCA), incubated for 5 minutes on ice, and subsequently extracted with ethanol:ethyl acetate (1:1, volume/volume), three times at 11000 ⫻ g for 10 minutes at 4oC. The pellets were carefully drained and dissolved in 6 M guanidine solution in H2O. The difference spectrum of the DNPH-treated sample versus the HCl control was determined at 360 nm, and the results expressed as nmol protein carbonyls/mg protein, using a molar extinction coefficient of 22,000 M⫺1 cm⫺1.25 Protein concentration was determined using the BioRad-Bradford assay.
Sigala et al 707
Data analysis and statistics The clinical samples were stratified as predefined groups of: 1) stable (noncomplicated) versus unstable (complicated) plaques; 2) symptomatic versus asymptomatic lesions; 3) the presence of ⱖ90% versus ⬍90% carotid stenosis. Differences in the levels of ox-LDL, NT, MDA, and PCs among all examined subgroups were estimated with the unpaired t test with Welch correction or the Mann-Whitney test/Kruskal-Wallis test (nonparametric analysis of variance) for variables with significant differences in their SDs, and their correlations were estimated by Pearson coefficient or Spearman nonparametric coefficient. The expressional relationship between the immunohistochemically (IHC) assessed indices of NT and MMP-9, within the predefined groups, were estimated by Pearson coefficient or Spearman nonparametric coefficient. All calculations were performed with the GraphPad Prism 4 software (GraphPad Software Inc). Values of P ⬍ .05 were considered statistically significant. RESULTS Increased ox-LDL levels in unstable and symptomatic carotid plaques We employed an ELISA assay to measure the ox-LDL levels directly in homogenates of tissue carotids. Analysis in our set of clinical samples showed a significant difference of ox-LDL levels only in unstable plaques in relation to either stable ones or the control group (P ⬍ .05, respectively; Fig 1A). A similar significant increase in ox-LDL levels was also found in symptomatic carotid lesions (P ⬍ .05; Fig 1B), but was not related to patients’ carotid stenosis (Fig 1C). NT levels increase in a stepwise manner in relation to plaque stability status ELISA assessment in tissue homogenates. Similarly as per ox-LDL, we assessed the NT status in the same tissue homogenates in relation to plaque stability, carotid stenosis, and symptomatic disease. NT levels increased significantly in a stepwise manner from the control group to the stable one (P ⬍ .05), and finally to the unstable one (P ⬍ .05; Fig 2A). Although, no difference was found in the NT levels regarding the status of the disease and of the carotid stenosis (Fig 2B and 2C, respectively), NT was increased in the patients stratified according to these parameters and compared with control group (P ⬍ .05, respectively; Fig 2B and 2C, respectively). Immunohistochemical analysis. Next we decided to examine the type, the topological distribution, and the percent of the cells expressing NT in the plaque, respectively. The immunohistochemical expression of NT was observed in the cytoplasm of vascular smooth muscle cells (VSMCs) and inflammatory cells (macrophages and foam cells) surrounding the lipid core (cap), as well as in the shoulders of the plaque (Fig 3). NT IHC expression ranged from 3% to 70% (average, 33.73% ⫾ 23.0%) in the cohort of patients.
708 Sigala et al
Fig 1. Oxidized LDL (ox-LDL) levels (ng/mL) in stable (noncomplicated) and unstable (complicated) plaques (A); symptomatic and asymptomatic lesions (B); ⱖ90% carotid stenosis and ⬍90% carotid stenosis (C). *P ⬍ .05 versus control samples, **P ⬍ .05 versus stable and asymptomatic.
NT immunostaining correlated with the ELISAassessed levels (P ⬍ .05; data not shown). MMP-9 expression correlates with NT in carotid plaques Data from cardiovascular models support that MMPs can be activated by oxidative stress. Some have shown that this activation is via intermediates of nitrosative stress,15-17 while others have shown an ox-LDL-dependence.18,19 To investigate this issue in vivo, we examined MMP-9 expression and searched for potential relationships with NT and ox-LDL, respectively, in our clinical setting. We choose MMP-9 as a representative MMP to study, as we8 and others26 have shown its significant increase in human ruptured carotid plaques. MMP-9 IHC analysis. MMP-9 IHC analysis showed its presence particularly in areas with intense inflammatory infiltration (presence of macrophages and foam cells), in VSMCs, and extracellular matrix (Fig 3). MMP-9 IHC expression ranged from 2% to 70% (average, 32.06% ⫾ 23.30%) in the clinical samples. A higher MMP-9 IHC LI was found in unstable plaques (35.26% ⫾ 22.57%) compared with stable ones (28.25% ⫾ 24.32%).
JOURNAL OF VASCULAR SURGERY September 2010
Fig 2. Nitrotyrosine (NT) levels (nmol/mg protein) in stable (noncomplicated) and unstable (complicated) plaques (A); symptomatic and asymptomatic lesions (B); ⱖ90% carotid stenosis and ⬍90% carotid stenosis (C). *P ⬍ .05 versus control samples, **P ⬍ .05 versus stable plaques.
Interestingly, an IHC analysis in serial sections showed that cells positive for NT immunohistochemical staining were topologically localized in areas coinciding with cells that exhibited MMP-9 IHC expression in both stable and unstable plaques (Fig 3). Notably, the IHC expression of these factors correlated (r ⫽ 0.98, P ⬍ .05; Fig 4). There was no association between MMP-9 expression and ox-LDL levels. Western blot analysis. In order to confirm that the higher MMP-9 IHC LIs in unstable plaques correspond to a quantitative increase in MMP-9 protein levels, we also performed Western blot analysis in representative randomly selected stable and unstable samples. As shown in Fig 5, MMP-9 expression levels were found significantly increased in unstable plaques in comparison to the stable ones (P ⬍ .05). Of note, both stable and unstable plaques also exhibited higher MMP-9 protein levels than control plaques (data not shown).
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
Sigala et al 709
Fig 3. Immunohistochemical (IHC) analysis of matrix metalloproteinase-9 (MMP-9) and nitrotyrosine (NT) in serial sections from stable and unstable plaques. MMP-9 and NT IHC co-localization in stable (A) and unstable (B) plaques at 200⫻ and 400⫻ magnification, respectively. Frames in 200⫻ magnification pictures represent the areas of 400⫻ magnification.
MDA and PCs are increased but not related to plaque destabilization and symptomatic disease Next, we extended our analysis to other markers of oxidative stress involved in atherogenesis10,20 and investigated for potential association(s) with plaque lesion destabilization. MDA and PCs levels were significantly increased in the patients’ cohort as compared with the control group (Figs 6 and
7). Nevertheless, there was no significant difference in MDA levels between stable and unstable plaques (P ⬎ .05). A similar result was also found between symptomatic and asymptomatic lesions (P ⬎ .05) as well as between patients with ⱖ90% and ⬍90% carotid stenosis (P ⬎ .05; Fig. 6). Finally, no significant difference was found between levels of PCs in stable and unstable plaques (P ⬎ .05), symptomatic
710 Sigala et al
JOURNAL OF VASCULAR SURGERY September 2010
Fig 4. Correlation between the immunohistochemical (IHC) labeling indices (LI) of matrix metalloproteinase-9 (MMP-9) and nitrotyrosine (NT) in the examined cohort of human carotid plaques (r ⫽ 0.98, P ⬍ .05).
Fig 6. Malondialdehyde (MDA) levels (mol/mg protein) in stable (noncomplicated) and unstable (complicated) plaques (A); symptomatic and asymptomatic lesions (B); ⱖ90% carotid stenosis and ⬍90% carotid stenosis (C). *P ⬍ .05 versus control samples.
Fig 5. Analysis of matrix metalloproteinase-9 (MMP-9) protein expression in stable and unstable plaques. Western blot analysis of MMP-9 expression in representative samples of stable and unstable plaques. (A); Histogram showing the significantly increased MMP-9 protein expression levels in unstable plaques, compared with stable ones (P ⬍ .05) (B).
and asymptomatic lesions (P ⬎ .05), as well as between patients with ⱖ90% and ⬍90% carotid stenosis (P ⬎ .05; Fig. 7). DISCUSSION Atherogenesis is a complex process of multifactorial etiology.5 Oxidative stress has been proposed as an important determinant in this process acting directly as well as mediating the associated inflammation with this pathologic
condition.6,27 Nevertheless, a systematic assessment of various established oxidative stress markers, especially in relation to plaque destabilization and rupture, is, to the best of our knowledge, missing. In this context, we performed a global investigation of four established markers of oxidative stress (such as ox-LDL, NT, MDA, and PCs) in a clinical setting of human carotid plaques, stratified according to their histologic status, degree of stenosis, and patients’ clinical status. The first marker we assessed was oxidized-LDL. Oxidation of LDL is known to participate in the initiation and progression of atherogenesis.10,11 Nevertheless, direct assessment of ox-LDL levels in tissue homogenates derived from carotid plaques, and a systematic correlation with classical clinicopathological parameters, such as plaque stability, carotid stenosis, and symptomatic disease, is scarce and incomplete.28-30 In our study, a first important finding we observed was the significantly increased ox-LDL levels in carotid lesions of symptomatic patients. This result was also corroborated by a corresponding increase in unstable
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
Fig 7. Protein carbonyls (PCs) levels (nmol/mg protein) in stable (noncomplicated) and unstable (complicated) plaques (A); symptomatic and asymptomatic lesions (B); ⱖ90% carotid stenosis and ⬍90% carotid stenosis (C). *P ⬍ .05 versus control samples.
plaques (type VI). Similar to our results, Uno et al observed an increase of plasma and plaque levels of ox-LDL in patients with vulnerable carotid plaques,29 suggesting that plasma ox-LDL assessment could be used as a potential marker for symptomatology development. The vascular wall ox-LDL has a crucial role in the development and progression of atherosclerosis11; however, its role in the mechanism of plaque instability and in the subsequent clinical events remains poorly understood. Pathways that oxidize lipids and proteins may thus be pivotal to the atherosclerotic process. ROS and RNS species mediate the oxidative modification of LDL.12 Oxidized LDL exerts several biological effects that may contribute to the progression and the disruption of the atherosclerotic lesions.4,31 It is a chemoattractant for monocytes and T lymphocytes, and it also inhibits macrophage mobility, thereby promoting retention of macrophages into the arterial wall.11 Oxidized LDL is cytotoxic and promotes endothelial dysfunction and the evolution of the fatty streak into a more advanced stage and unstable lesion.32 It can also accelerate atherogenesis by stimulating the expression of
Sigala et al 711
several other genes into the arterial wall, such as interleukin-1. In addition, ox-LDL can adversely affect coagulation by stimulating tissue factor and plasminogen activator inhibitor-1 synthesis.33 Ox-LDL inhibits endotheliumderived relaxation factor-mediated vasodilation.34 Overexpression of oxidative LDL levels is an active stimulation of the inflammation that occurs into the atherosclerotic plaque, thus promoting the instability of the plaque and the manifestation of clinical events. However, the identity and the sources of the various oxidants that initiate and produce LDL oxidation in artery carotid disease have not yet been clearly defined. Nitrosative stress may be a possible mechanism by which plaque instability is promoted through ox-LDL production.15,16 In this context, our results showing a significant stepwise increase in NT levels specifically from stable to unstable plaques are suggestive. The production of NO by inducible nitric oxide synthase, in the presence of superoxide anion produced by macrophages, results in the formation of peroxynitrite.14 Several lines of evidence indicate that nitrotyrosine levels are not only detrimental for the development of atherosclerosis,35-37 but are also linked with oxidation of LDL.6,12,13 The absence of association between ox-LDL and NT levels in our cohort of patients implies that other mechanisms may also be responsible for oxidation of LDL. Of note, several cardiovascular models support that MMPs can be activated by oxidative stress. Some have shown that this activation is via intermediates of nitrosative stress,15-17 while others support an ox-LDL-dependence.18,19 Our IHC analysis in serial sections showed a co-localization of NT and MMP-9 that was also supported by a significant statistical expressional correlation. The expression of MMP-9 not only promotes the degradation of extracellular matrix, but also induces the migration and proliferation of smooth muscle cells.26,38 In association with endothelial cell basement membrane degradation and enhanced endothelial cell permeability, this results in greater influx of plasma constituents including lipoproteins, thereby accelerating plaque formation and instability. In our study, we showed a significant association between NT and MMP-9 expression for the first time in human carotid plaques, suggesting that peroxynitrite probably decreases the integrity of the connective network, leading to a decrease in plaque stability and, therefore, facilitating plaque rupture. Two other markers examined in this study are MDA and PCs. MDA is a highly reactive dialdehyde and represents an alternative route of LDL oxidation,38,39 while carbonyl groups (aldehydes and ketones) produced on protein side chains after oxidation represent an established marker of protein oxidation.40 In our study, we observed significantly increased MDA and PCs levels in atherosclerotic carotid plaques compared with normal lesions, but there were no significant differences between the clinical samples stratified according to the clinicopathologic parameters employed. Our data suggest that the lipid peroxidation process, by increased MDA plaque production and protein oxidation, probably contribute to plaque progres-
712 Sigala et al
sion, mainly at early stages, but it seems that they are not associated with latter stages related to plaque vulnerability and disruption. In conclusion, our findings support that oxidative stress is an important modulator of atherosclerosis. Specifically, our observations point at ox-LDL and peroxynitrite as important markers associated with advanced stages of this disease, concerning plaque destabilization and rupture. These findings could be exploited in the future in the form of potential markers that could be used to assist in prevention and therapeutic decision. AUTHOR CONTRIBUTIONS Conception and design: FS, AK, VG, IA Analysis and interpretation: AK, PS, KF, VG, IA Data collection: FS, KF Writing the article: FS, AK Critical revision of the article: FS, AK, EI, VG, IA Final approval of the article: FS, AK, PS, KF, SM, EI, VG, IA Statistical analysis: SM, EI Obtained funding: FS, AK, VG, IA Overall responsibility: VG FS and AK contributed equally to this work. REFERENCES 1. Yusuf S, Reddy S, Onupuus S, Anand S. Global burden of cardiovascular diseases: Part 1: General considerations, the epidemiologic transition, risk factors and impact of urbanization. Circulation 2001;104:2746-53. 2. Sacco RL, Benjamin EJ, Broderick JP, Dyken M, Easton JD, Feinberg WM, et al. American Heart Association Prevention Conference. IV. Prevention and Rehabilitation of Stroke. Risk factors. Stroke 1997;28: 1507-17. 3. North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. N Engl J Med 1991;325:445-53. 4. Aikawa M, Libby P. The vulnerable atherosclerotic plaque: pathogenesis and therapeutic approach. Cardiovasc Pathol 2004;1:125-38. 5. Koenig W, Khusevinova N. Biomarkers of atherosclerotic plaque instability and rupture. Arterioscler Thromb Biol 2007;27:15-26. 6. Lamon BD, Haijar DP. Inflammation at the molecular interface of atherogenesis: an anthropological journey. Am J Pathol 2008;173: 1253-64. 7. Sigala F, Georgopoulos S, Papalambros E, Chasiotis D, Vourliotaki G, Niforou A, et al. Heregulin, cysteine rich-61 and matrix metalloproteinase 9 expression in human carotid atherosclerotic plaques: relationship with clinical data. Eur J Vasc Endovasc Surg 2006;32:238-5. 8. Papalambros E, Sigala F, Georgopoulos S, Panou N, Kavantzas N, Agapitos M, et al. Vascular endothelial growth factor and matrix metalloproteinase-9 expression in human carotid atherosclerotic plaques: relationship with plaque destabilization via neovascularization. Cerebrovasc Dis 2004;18:160-5. 9. Schulze PC, Lee RT. Oxidative stress and atherosclerosis. Curr Atheroscler Rep 2005;7:242-8. 10. Parthasarathy S, Steinberg D, Witztum JL. The role of oxidized lowdensity lipoproteins in the pathogenesis of atherosclerosis. Annu Rev Med 1992;43:219-25. 11. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 1997;272:20963-6. 12. Napoli C, De Nigris F, Palinski W. Multiple role of reactive oxygen species in the arterial wall. J Cell Biochem 2001;82:674-82. 13. Matsunaga T. Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins. J Biochem 2001;130:285-93.
JOURNAL OF VASCULAR SURGERY September 2010
14. Reiters CD, Teng RJ, Eckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 2000;270:32460-6. 15. Rajagopalan S, Meng XP, Ramasamy S, Harrison DG, Glis ZS. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. J Clin Invest 1996;98:2572-9. 16. Egi K, Conrad N, Kwan J, Schulze C, Schulz R, Wildhirt SM. Inhibition of the inducible nitric oxide synthase and superoxide production reduces matrix metalloproteinase-9 activity and restores coronary vasomotor function in rat cardiac allografts. Eur J Cardiothorac Surg 2004;26:262-9. 17. Mu H, Wang X, Lin P, Yao Q, Chen C. Nitrotyrosine promotes human aortic smooth muscle cell migration throughoxidative stress and ERK1/2 activation. Biochim Biophys Acta 2008;1783:1576-84. 18. Huang Y, Mironova M, Lopes-Virella MF. Oxidized LDL stimulates matrix metalloproteinase-1 expression in human vascular endothelial cells. Arterioscler Thromb Vasc Biol 1999;19:2640-7. 19. Xu XP, Meisel SR, Ong JM, Kaul S, Cercek B, Rajavashisth TB. Oxidized low-density lipoprotein regulates matrix metalloproteinase-9 and its tissue inhibitor in human monocyte-derived macrophages. Circulation 1999;99:993-8. 20. Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 1997;272:20313-6. 21. Hernandez E, Goel N, Dougherty KG, Strickman NE, Krajcer Z. Benefits of catheter thrombectomy during carotid stenting. Tex Heart Inst J 2009;36:404-8. 22. Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W Jr, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 1995;92:1355-74. 23. Gorgoulis VG, Pratsinis H, Zacharatos P, Demoliou C, Sigala F, Asimacopoulos PA, et al. p53-Dependent ICAM-1 overexpression in senescent human cells identified in atherosclerotic lesions. Lab Invest 2005;85:502-11. 24. Andreadou I, Iliodromitis EK, Tsovolas K, Aggeli IK, Zoga A, Gaitanaki C, et al. Acute administration of Vitamin E triggers preconditioning via KATP channels and cyclic-GMP without inhibiting lipid peroxidation. Free Radic Biol Med 2006;41:1092-9. 25. Andreadou I, Iliodromitis EK, Mikros E, Constantinou M, Agalias A, Magiatis P, et al. The olive constituent oleuropein exhibits antiischemic, anti-oxidative and hypolipidemic effects in anesthetized rabbits. J Nutr 2006;136:2213-9. 26. Loftus IM, Naylor AR, Bell PR, Thompson MM. Matrix metalloproteinases and atherosclerotic plaque instability. Br J Surg 2002;89: 680-94. 27. Stocker R, Keaney JF Jr. New insights on oxidative stress in the artery wall. J Thromb Haemost 2005;3:1825-34. 28. Nishi K, Itabe H, Uno M, Kitazato KT, Horiguchi H, Shinno K, Nagahiro S. Oxidized LDL in carotid plaques and plasma associates with plaque instability. Arterioscler Thromb Vasc Biol 2002;22: 1649-54. 29. Uno M, Kitazato KT, Suzue A, Itabe H, Hao L, Nagahiro S. Contribution of an imbalance between oxidant-antioxidant systems to plaque vulnerability in patients with carotid artery stenosis. J Neurosurg 2005; 103:518-22. 30. Mannheim D, Herrmann JH, Versari D, Gössl M, Meyer FB, McConnell JP, et al. Enhanced expression of LP-PLA2 and lysophosphatidylcholine in symptomatic carotid atherosclerotic plaques. Stroke 2008; 39:1448-55. 31. Quehenberger O. Molecular mechanisms regulating monocyte recruitment in atherosclerosis. J Lipid Res 2005;46:1582-90. 32. JialalI I. Evolving lipoprotein risk factors: lipoprotein(a) and oxidized low-density lipoprotein. Clin Chem 1998;44:1827-32. 33. Clinton SK, Libby P. Cytokines and growth factors in atherogenesis. Arch Path Lab Med 1992;116:1292-300. 34. Clinton I. Oxidized low density lipoprotein and atherosclerosis. Int J Clin Lab Res 1996;26:178-84.
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
Sigala et al 713
35. Leeuwenburhg C, Hardy MM, Hazen SL, Wagner P, Ohishi S, Steinbrecher UP, et al. Reactive nitrogen Intermediates promote low density lipoprotein Oxidation in human atherosclerotic intima. J Biol Chem 1997;272:1433-6. 36. Hunter GC, Henderson AM, Westerbamd A, Kobayashi H, Suzuki F, Yan Z, et al. The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque. J Vasc Surg 1999;30:36-50. 37. Loftus IM, Naylor AR, Goodall S, Crowther M, Jones L, Bell PR, Thompson MM. Increased matrix metalloproteinase activity in unstable carotid plaques. A potential role in acute plaque disruption. Stroke 2005;31:40-47.
38. Ross R. The pathogenesis of atherosclerosis. A perspective for the 1990s. Nature 1993;362:801-9. 39. Minuz P, Fava C, Lechi A. Lipid peroxidation, isoprostanes and vascular damage. Pharmacol Rep 2006;57:57-68. 40. Dalle-Done I, Rossi R, Giustarini D, Milzani A, Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 2003;329:23-38.
Submitted Dec 18, 2009; accepted Mar 20, 2010.
AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of Journal of Vascular Surgery for the preceding 5 years are maintained and are available for purchase from Mosby until inventory is depleted. Please write to Elsevier Inc., Subscription Customer Service, 11830 Westline Industrial Drive, St. Louis, MO, or call 800-654-2452 (U.S. and Canada); 314-453-7041 (outside U.S. and Canada) for information on availability of particular issues and prices.
Cardiovascular mortality is the major cause of death in adults older than 65 years in Western societies.1 AtheroscleFrom the First Department of Propaedeutic Surgery, Hippocrateion Hospital, Medical School, University of Athens,a the Molecular Carcinogenesis Group, Department of Histology-Embryology, Medical School, University of Athens,b the Laboratory of Biopharmaceutics and Pharmacokineticsc and the Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens,d the Second University Department of Cardiology, Medical School, University of Athens, Attikon Hospital.e Supported by National Kapodistrian University of Athens-Special Account for Research Grants (NKUA-SARG) grants No 70/4/9926, 70/4/ 9900, 70/4/9913, and 70/4/4281. VGG is also supported by the European Commission Grant FP7 INFLA-CARE (NKUA-SARG code 70/3/9815). Competition of interest: none. Reprint requests: Vassilis G. Gorgoulis, Antaiou 53 Str., Lamprini, Ano Patissa, GR-11146 Athens, Greece 15771 (e-mail: [email protected]) and Ioanna Andreadou, Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimioupolis, Zografou, Athens, Greece (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a competition of interest. 0741-5214/$36.00 Copyright © 2010 by the Society for Vascular Surgery. doi:10.1016/j.jvs.2010.03.047
704
rotic disease of the carotid artery is responsible for 20% to 30% of all strokes.2 At present, the only established criterion for predicting stroke is the degree of carotid stenosis.3 It seems, however, that plaque instability is an essential determinant of clinical manifestation of symptomatic carotid occlusive disease.4 The remodeling of a stable asymptomatic lesion into an unstable, ruptured plaque is a complex process.5,6 A plethora of data5,7,8 have implicated a variety of factors in plaque destabilization and eventually plaque rupture. Increasing evidence has shown that inflammation has a crucial role in the cascade of events related to plaque erosion.5,6 However, the molecular mechanisms that induce inflammation and subsequent plaque instability have not yet been completely deciphered. Available data have pointed to, among other mechanisms, the oxidative stress in these processes.9 Oxidation of low-density lipoprotein (LDL) has been shown to be a key factor for the atherogenetic development.10,11 Various reactive intermediates, derivatives of reactive oxygen (ROS) and/or reactive nitrogen (RNS) species, are known to mediate the oxidative modification of LDL.6,12,13 Among
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
Sigala et al 705
Table. Patients’ demographics and clinicopathological data Patients Demographic data Number Mean age (range) Male/female Smoking Current/past smokers Clinical data Hypertension Diabetes Hyperlipidemia Ischemic heart disease Aneurysm Peripheral arterial occlusive disease Clinical symptoms Stroke Transient ischemic attack Amaurosis fugax Angiographic carotic stenosis ⬍90% ⱖ90% Computed tomography brain (positive/negative) Plaque histopathology status Unstable Stable Medications Statins Angiotensin-converting enzyme inhibitors -blockers
Overall
Symptomatic
Asymptomatic
36 67.05 (55-80) 31/5 28 12/16
19 67.2 16/3 16 9/7
17 66.9 15/2 12 5/7
31 10 18 20 3 11 19 6 9 4
15 5 10 10 2 7 19 6 9 4
16 5 8 10 1 4 — — — —
16 20 19/17
7 11 19/0
17 19
14 3
3 16
18 15 19
10 7 10
8 8 9
them, peroxynitrite, the reaction product between nitric oxide (NO) and superoxide anion radicals,14 and malondialdehyde, a product from lipid peroxidation,10 are powerful LDL oxidants, which generate modified LDL with increased atherogenic potential.10,11 Interestingly, oxidizedLDL (ox-LDL) can lead to further production of ROS, thus creating a sustained oxidative stress condition.6 Of note, activation of matrix metalloproteinases (MMPs) has been shown to contribute also to plaque destabilization, through extracellular matrix remodelling.5,7 In certain cardiovascular experimental models, this activation was nitrosative stress-intermediates dependent,15-17 while in others, ox-LDL has been shown to be responsible for their expression.18,19 Other forms of oxidative stress, such as lipid peroxidation and protein oxidation, have been described in atherogenesis,10,20 but their contribution in this process, and specifically in symptomatic carotid disease and plaque instability generation, requires further investigation. To the best of our knowledge, a global assessment of various oxidative stress markers directly in human carotid plaques stratified according to their histologic status, degree of stenosis, and patients’ clinical status, is missing. Therefore, in the present study, we first sought to collectively determine the levels of various biochemical markers related to oxidative stress in the same clinical setting of human carotid plaques. These markers included ox-LDL, along with nitrotyrosine (NT) - the fingerprint of peroxynitrite, malondialdehyde (MDA), and protein carbonyls
9 0 0/17
(PCs), as indicators of nitrosative stress, lipid peroxidation, and protein oxidation, respectively. Subsequently, we looked into possible association of these markers with expression of MMP-9 and plaque destabilization in human carotid artery disease. MATERIALS AND METHODS Tissue collection Patients. Between 2007 and 2009, carotid plaques were prospectively collected from 36 random patients, who had internal carotid artery stenosis ⬎70% and underwent carotid endarterectomy. Demographic data, clinicopathological data, medication, risk factors, and vascular comorbidities were recorded (Table). All patients were evaluated preoperatively by a neurologist to be assigned as symptomatic and asymptomatic. Symptomatic patients were classified based on the presence of stroke, transient ischemic attacks, and amaurosis fugax. They also underwent a cerebral CT scan for identification of brain infarcts. Arteriography of the carotid bifurcation was performed in all patients for this study. Degree of stenosis was determined according to North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria.3 Based on these measurements, stenotic lesions were divided into two subgroups (70%-90% and ⱖ90%-99%), as previously described.21 The study protocol was approved by the Institutional Ethics Committee and all patients enrolled gave their informed consent.
JOURNAL OF VASCULAR SURGERY September 2010
706 Sigala et al
Control tissues. Twenty healthy carotid arteries obtained from autopsies served as control tissue samples for the “baseline-expression” of the examined factors in human arteries. Criteria for their selection included no previous history of cardiovascular disease and cause of death unrelated to pathologic conditions (eg, accidents), and were matched by gender (18 males and 2 women) and age (mean, 65.8 years; range, 50-74 years) to the patients’ specimens under investigation. Tissue preparation All carotid plaque specimens were removed in the operating room and were divided into two portions. One portion was fixed immediately in 10% neutral-buffered solution with 4% formaldehyde for 24 hours, and embedded in paraffin. The second portion was immediately stored at ⫺70oC for further analysis of ox-LDL, MDA, NT, and PCs and Western-blot analysis. Histology Hematoxylin and eosin staining was performed for histological evaluation of the specimens. Two pathologists, blinded to the clinical data, examined each specimen to assess atheromatous plaque morphology, using the American Heart Association classification of atherosclerotic plaques.22 According to this classification, carotid plaques were assigned as fibroatherotic (type V) and complicated (type VI). The latter type included plaques with intraplaque hemorrhage, ulcer, or thrombus, which were considered unstable. Immunohistochemistry Antibodies. For immunohistochemical analysis the following antibodies were used: anti-Nitrotyrosine (HM11; Zymed, Anti-Sel, Athens, Greece) at a 1:50 dilution, MMP-9 sheep polyclonal antibody (SantaCruz Biogenesis, Greece), in a 1:100 dilution. In serial sections, we also determined the cellular phenotype of atherosclerotic plaques. To identify macrophages/foam cells, smooth muscle cells, and endothelial cells, the following mouse monoclonal antibodies were used at a 1:50 dilution, respectively: CD68 (KP1; Dako, Kalifronas, Greece), alphasmooth muscle actin (1A4; Dako), and CD34 mouse (QBE nd/10; Biogenex, Aenorasis, Athens, Greece).7,23 Method. Immunohistochemistry was performed according to the indirect streptavidin-biotin-peroxidase method. In brief, 5 m paraffin sections were placed on poly-L-lysine-coated slides, dewaxed, rehydrated, and incubated for 30 minutes with hydrogen peroxide 0.3% to quench the endogenous peroxidase activity. Unmasking of the related proteins was carried out. The sections were incubated with the primary antibody at 4°C overnight. Biotin-conjugated secondary antibody was added at 1:200 dilution for 1 hour at room temperature (RT). The next stage comprised 30 minutes incubation in StreptAB Complex (1:100 stock biotin solution, 1:100 stock streptavidinhyperoxidase solution; Dako). For color development, we used 3,3=-diaminobenzidine tetrahydrochloride (DAB,
Sigma-Hellas, Athens, Greece) and hematoxylin as a counterstain. Evaluation. The staining pattern was considered positive only if cytoplasmic signal was discerned. Images were obtained with a Zeiss-Axiolab microscope (Carl Zeiss GmbH, Jena, Germany), employing a video analysis software as previously described.8 The NT and MMP-9 labeling indices (LI) were determined as the percentage of cells with cytoplasmic immunopositivity, irrespective of staining intensity. Of note, MMP-9 extracellular matrix staining was also observed and considered specific, but was not estimated in terms of a LI. Two independent observers (AK and VG) carried out slide examination. Inter-observer variability was minimal. Immunoblotting Protein extraction. Total protein extraction was performed according to the protocol described elsewhere.7 Briefly, tissues were homogenized in 10 mM TrisHCl pH 8, 170 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 0.5% nonidet P-40, 1 mM dithiothreitol and centrifuged at 1000 ⫻ g at 4°C for 5 minutes. The supernatant was collected and adjusted to 1g/mL protease and phosphatase inhibitors (Sigma-Hellas). Total protein concentration was calculated using the Roti-Quant Bradford-Assay (Roth, BioSure, Athens, Greece). SDS-PAGE. Thirty g of protein from total extracts of each sample were adjusted with NuPAGE LDS Sample Buffer (Invitrogen, Anti-Sel, Athens, Greece) and loaded on 4% to 12% gradient NuPAGE precast gels (Invitrogen). Gel electrophoresis and transfer to polyvinylidene difluoride membrane (Millipore, AlterChem, Athens, Greece) were performed according to standard protocols. Blots were blocked overnight in 2.5% gelatin solution in Trisbuffered saline (Sigma-Hellas) at room temperature. Subsequently, membranes were incubated for 3 hours with primary antibody solution in Tris-buffered saline supplemented with 0.5% Tween-20 at RT, followed by a 45 minute incubation with HRP-conjugated anti-mouse secondary antibody (1:2500 dilution; R&D Systems, Anti-Sel, Athens, Greece) at RT. Signal development was performed with the Western Glo-Chemiluminescent Detection Substrate (R&D Systems).7,23 Antibodies. The antibodies used were: anti-MMP-9 (1:500; sheep polyclonal, Neomarkers, BioAnalytica, Athens, Greece) and anti-tubulin (1:1000; AC-15, Abcam, Anti-Sel, Athens, Greece) for assessing equal loading of total protein and for normalizing the MMP-9 levels. Ox-LDL determination Ox-LDL was determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturers’ instructions (Biomedica, Biomedica Gruppe, Vienna, Austria). The detection limit of the assay was 0.8 ng/mL. NT determination NT, the fingerprint of peroxynitrite in the carotid plaques, was determined by ELISA according to the man-
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
ufacturers’ instructions (Bioxytech, Nitrotyrosine-EIA; Oxis Research, Beverly Hills, Calif). The detection limit of the assay was 2 nM. Measurement of carotid MDA Human carotid samples were frozen at ⫺70oC until the assay. On analysis, tissue samples were washed in ice-cold NaCl 0.9%, blotted on absorbent paper, and weighed. Each sample was minced in ice-cold 20 mM Tris-HCl buffer pH 7.4, in a 1:10 weight/volume ratio, and homogenized using a Teflon pestle. After centrifugation at 3000 ⫻ g for 10 minutes at 4oC, the supernatant was used for the biochemical assay.24 MDA concentration was determined spectrophotometrically at 586 nm and expressed in M (Oxford Biomedical Research Colorimetric Assay for lipid peroxidation) with some modifications.23 The method used was based on the reaction of the chromogenic reagent N-methyl2-phenylindole with MDA. One molecule of free MDA combines with two molecules of the chromogenic agent to produce a stable chromophore with a maximal absorbance at 586 nm. Briefly, 0.65 mL of 10.3 mM N-methyl-2-phenylindole in acetonitrile was added to 0.2 mL of tissue homogenate. After vortexing for 3 to 4 seconds and adding 0.15 mL of HCl 37%, samples were mixed well, closed with a tight stopper, and incubated at 45o C for 60 minutes. The samples were then cooled on ice, centrifuged, and the absorbance was measured spectrophotometrically at 586 nm. A calibration curve, comprising accurately prepared standard MDA solutions (from 2 to 20 nmol/mL), was also run for quantitation. Measurements of each group were performed in triplicate. Determination of protein carbonyls content A small amount of tissue was rinsed with phosphatebuffer saline solution to remove red blood cells or clots. Tissue was homogenized in 1 mL of cold phosphate-buffer pH 6.7, containing 1 mM EDTA, and then centrifuged at 10,000 ⫻ g for 15 minutes at 4oC. The supernatant was removed, and the tissue was stored at ⫺80oC until use. A modification of the technique of Levine et al,25 based on spectrophotometric measurement of 2,4-dinitrophenylhydrazine (DNPH) derivatives of protein carbonyls, was used to quantify proteincarbonyl content. Briefly, 100 L of plasma was incubated either with 500 L DNPH or 2 N HCl (blank) for 1 hour at RT. The samples were then reprecipated with 600 l 20% trichloroacetic acid (TCA), incubated for 5 minutes on ice, and subsequently extracted with ethanol:ethyl acetate (1:1, volume/volume), three times at 11000 ⫻ g for 10 minutes at 4oC. The pellets were carefully drained and dissolved in 6 M guanidine solution in H2O. The difference spectrum of the DNPH-treated sample versus the HCl control was determined at 360 nm, and the results expressed as nmol protein carbonyls/mg protein, using a molar extinction coefficient of 22,000 M⫺1 cm⫺1.25 Protein concentration was determined using the BioRad-Bradford assay.
Sigala et al 707
Data analysis and statistics The clinical samples were stratified as predefined groups of: 1) stable (noncomplicated) versus unstable (complicated) plaques; 2) symptomatic versus asymptomatic lesions; 3) the presence of ⱖ90% versus ⬍90% carotid stenosis. Differences in the levels of ox-LDL, NT, MDA, and PCs among all examined subgroups were estimated with the unpaired t test with Welch correction or the Mann-Whitney test/Kruskal-Wallis test (nonparametric analysis of variance) for variables with significant differences in their SDs, and their correlations were estimated by Pearson coefficient or Spearman nonparametric coefficient. The expressional relationship between the immunohistochemically (IHC) assessed indices of NT and MMP-9, within the predefined groups, were estimated by Pearson coefficient or Spearman nonparametric coefficient. All calculations were performed with the GraphPad Prism 4 software (GraphPad Software Inc). Values of P ⬍ .05 were considered statistically significant. RESULTS Increased ox-LDL levels in unstable and symptomatic carotid plaques We employed an ELISA assay to measure the ox-LDL levels directly in homogenates of tissue carotids. Analysis in our set of clinical samples showed a significant difference of ox-LDL levels only in unstable plaques in relation to either stable ones or the control group (P ⬍ .05, respectively; Fig 1A). A similar significant increase in ox-LDL levels was also found in symptomatic carotid lesions (P ⬍ .05; Fig 1B), but was not related to patients’ carotid stenosis (Fig 1C). NT levels increase in a stepwise manner in relation to plaque stability status ELISA assessment in tissue homogenates. Similarly as per ox-LDL, we assessed the NT status in the same tissue homogenates in relation to plaque stability, carotid stenosis, and symptomatic disease. NT levels increased significantly in a stepwise manner from the control group to the stable one (P ⬍ .05), and finally to the unstable one (P ⬍ .05; Fig 2A). Although, no difference was found in the NT levels regarding the status of the disease and of the carotid stenosis (Fig 2B and 2C, respectively), NT was increased in the patients stratified according to these parameters and compared with control group (P ⬍ .05, respectively; Fig 2B and 2C, respectively). Immunohistochemical analysis. Next we decided to examine the type, the topological distribution, and the percent of the cells expressing NT in the plaque, respectively. The immunohistochemical expression of NT was observed in the cytoplasm of vascular smooth muscle cells (VSMCs) and inflammatory cells (macrophages and foam cells) surrounding the lipid core (cap), as well as in the shoulders of the plaque (Fig 3). NT IHC expression ranged from 3% to 70% (average, 33.73% ⫾ 23.0%) in the cohort of patients.
708 Sigala et al
Fig 1. Oxidized LDL (ox-LDL) levels (ng/mL) in stable (noncomplicated) and unstable (complicated) plaques (A); symptomatic and asymptomatic lesions (B); ⱖ90% carotid stenosis and ⬍90% carotid stenosis (C). *P ⬍ .05 versus control samples, **P ⬍ .05 versus stable and asymptomatic.
NT immunostaining correlated with the ELISAassessed levels (P ⬍ .05; data not shown). MMP-9 expression correlates with NT in carotid plaques Data from cardiovascular models support that MMPs can be activated by oxidative stress. Some have shown that this activation is via intermediates of nitrosative stress,15-17 while others have shown an ox-LDL-dependence.18,19 To investigate this issue in vivo, we examined MMP-9 expression and searched for potential relationships with NT and ox-LDL, respectively, in our clinical setting. We choose MMP-9 as a representative MMP to study, as we8 and others26 have shown its significant increase in human ruptured carotid plaques. MMP-9 IHC analysis. MMP-9 IHC analysis showed its presence particularly in areas with intense inflammatory infiltration (presence of macrophages and foam cells), in VSMCs, and extracellular matrix (Fig 3). MMP-9 IHC expression ranged from 2% to 70% (average, 32.06% ⫾ 23.30%) in the clinical samples. A higher MMP-9 IHC LI was found in unstable plaques (35.26% ⫾ 22.57%) compared with stable ones (28.25% ⫾ 24.32%).
JOURNAL OF VASCULAR SURGERY September 2010
Fig 2. Nitrotyrosine (NT) levels (nmol/mg protein) in stable (noncomplicated) and unstable (complicated) plaques (A); symptomatic and asymptomatic lesions (B); ⱖ90% carotid stenosis and ⬍90% carotid stenosis (C). *P ⬍ .05 versus control samples, **P ⬍ .05 versus stable plaques.
Interestingly, an IHC analysis in serial sections showed that cells positive for NT immunohistochemical staining were topologically localized in areas coinciding with cells that exhibited MMP-9 IHC expression in both stable and unstable plaques (Fig 3). Notably, the IHC expression of these factors correlated (r ⫽ 0.98, P ⬍ .05; Fig 4). There was no association between MMP-9 expression and ox-LDL levels. Western blot analysis. In order to confirm that the higher MMP-9 IHC LIs in unstable plaques correspond to a quantitative increase in MMP-9 protein levels, we also performed Western blot analysis in representative randomly selected stable and unstable samples. As shown in Fig 5, MMP-9 expression levels were found significantly increased in unstable plaques in comparison to the stable ones (P ⬍ .05). Of note, both stable and unstable plaques also exhibited higher MMP-9 protein levels than control plaques (data not shown).
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
Sigala et al 709
Fig 3. Immunohistochemical (IHC) analysis of matrix metalloproteinase-9 (MMP-9) and nitrotyrosine (NT) in serial sections from stable and unstable plaques. MMP-9 and NT IHC co-localization in stable (A) and unstable (B) plaques at 200⫻ and 400⫻ magnification, respectively. Frames in 200⫻ magnification pictures represent the areas of 400⫻ magnification.
MDA and PCs are increased but not related to plaque destabilization and symptomatic disease Next, we extended our analysis to other markers of oxidative stress involved in atherogenesis10,20 and investigated for potential association(s) with plaque lesion destabilization. MDA and PCs levels were significantly increased in the patients’ cohort as compared with the control group (Figs 6 and
7). Nevertheless, there was no significant difference in MDA levels between stable and unstable plaques (P ⬎ .05). A similar result was also found between symptomatic and asymptomatic lesions (P ⬎ .05) as well as between patients with ⱖ90% and ⬍90% carotid stenosis (P ⬎ .05; Fig. 6). Finally, no significant difference was found between levels of PCs in stable and unstable plaques (P ⬎ .05), symptomatic
710 Sigala et al
JOURNAL OF VASCULAR SURGERY September 2010
Fig 4. Correlation between the immunohistochemical (IHC) labeling indices (LI) of matrix metalloproteinase-9 (MMP-9) and nitrotyrosine (NT) in the examined cohort of human carotid plaques (r ⫽ 0.98, P ⬍ .05).
Fig 6. Malondialdehyde (MDA) levels (mol/mg protein) in stable (noncomplicated) and unstable (complicated) plaques (A); symptomatic and asymptomatic lesions (B); ⱖ90% carotid stenosis and ⬍90% carotid stenosis (C). *P ⬍ .05 versus control samples.
Fig 5. Analysis of matrix metalloproteinase-9 (MMP-9) protein expression in stable and unstable plaques. Western blot analysis of MMP-9 expression in representative samples of stable and unstable plaques. (A); Histogram showing the significantly increased MMP-9 protein expression levels in unstable plaques, compared with stable ones (P ⬍ .05) (B).
and asymptomatic lesions (P ⬎ .05), as well as between patients with ⱖ90% and ⬍90% carotid stenosis (P ⬎ .05; Fig. 7). DISCUSSION Atherogenesis is a complex process of multifactorial etiology.5 Oxidative stress has been proposed as an important determinant in this process acting directly as well as mediating the associated inflammation with this pathologic
condition.6,27 Nevertheless, a systematic assessment of various established oxidative stress markers, especially in relation to plaque destabilization and rupture, is, to the best of our knowledge, missing. In this context, we performed a global investigation of four established markers of oxidative stress (such as ox-LDL, NT, MDA, and PCs) in a clinical setting of human carotid plaques, stratified according to their histologic status, degree of stenosis, and patients’ clinical status. The first marker we assessed was oxidized-LDL. Oxidation of LDL is known to participate in the initiation and progression of atherogenesis.10,11 Nevertheless, direct assessment of ox-LDL levels in tissue homogenates derived from carotid plaques, and a systematic correlation with classical clinicopathological parameters, such as plaque stability, carotid stenosis, and symptomatic disease, is scarce and incomplete.28-30 In our study, a first important finding we observed was the significantly increased ox-LDL levels in carotid lesions of symptomatic patients. This result was also corroborated by a corresponding increase in unstable
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
Fig 7. Protein carbonyls (PCs) levels (nmol/mg protein) in stable (noncomplicated) and unstable (complicated) plaques (A); symptomatic and asymptomatic lesions (B); ⱖ90% carotid stenosis and ⬍90% carotid stenosis (C). *P ⬍ .05 versus control samples.
plaques (type VI). Similar to our results, Uno et al observed an increase of plasma and plaque levels of ox-LDL in patients with vulnerable carotid plaques,29 suggesting that plasma ox-LDL assessment could be used as a potential marker for symptomatology development. The vascular wall ox-LDL has a crucial role in the development and progression of atherosclerosis11; however, its role in the mechanism of plaque instability and in the subsequent clinical events remains poorly understood. Pathways that oxidize lipids and proteins may thus be pivotal to the atherosclerotic process. ROS and RNS species mediate the oxidative modification of LDL.12 Oxidized LDL exerts several biological effects that may contribute to the progression and the disruption of the atherosclerotic lesions.4,31 It is a chemoattractant for monocytes and T lymphocytes, and it also inhibits macrophage mobility, thereby promoting retention of macrophages into the arterial wall.11 Oxidized LDL is cytotoxic and promotes endothelial dysfunction and the evolution of the fatty streak into a more advanced stage and unstable lesion.32 It can also accelerate atherogenesis by stimulating the expression of
Sigala et al 711
several other genes into the arterial wall, such as interleukin-1. In addition, ox-LDL can adversely affect coagulation by stimulating tissue factor and plasminogen activator inhibitor-1 synthesis.33 Ox-LDL inhibits endotheliumderived relaxation factor-mediated vasodilation.34 Overexpression of oxidative LDL levels is an active stimulation of the inflammation that occurs into the atherosclerotic plaque, thus promoting the instability of the plaque and the manifestation of clinical events. However, the identity and the sources of the various oxidants that initiate and produce LDL oxidation in artery carotid disease have not yet been clearly defined. Nitrosative stress may be a possible mechanism by which plaque instability is promoted through ox-LDL production.15,16 In this context, our results showing a significant stepwise increase in NT levels specifically from stable to unstable plaques are suggestive. The production of NO by inducible nitric oxide synthase, in the presence of superoxide anion produced by macrophages, results in the formation of peroxynitrite.14 Several lines of evidence indicate that nitrotyrosine levels are not only detrimental for the development of atherosclerosis,35-37 but are also linked with oxidation of LDL.6,12,13 The absence of association between ox-LDL and NT levels in our cohort of patients implies that other mechanisms may also be responsible for oxidation of LDL. Of note, several cardiovascular models support that MMPs can be activated by oxidative stress. Some have shown that this activation is via intermediates of nitrosative stress,15-17 while others support an ox-LDL-dependence.18,19 Our IHC analysis in serial sections showed a co-localization of NT and MMP-9 that was also supported by a significant statistical expressional correlation. The expression of MMP-9 not only promotes the degradation of extracellular matrix, but also induces the migration and proliferation of smooth muscle cells.26,38 In association with endothelial cell basement membrane degradation and enhanced endothelial cell permeability, this results in greater influx of plasma constituents including lipoproteins, thereby accelerating plaque formation and instability. In our study, we showed a significant association between NT and MMP-9 expression for the first time in human carotid plaques, suggesting that peroxynitrite probably decreases the integrity of the connective network, leading to a decrease in plaque stability and, therefore, facilitating plaque rupture. Two other markers examined in this study are MDA and PCs. MDA is a highly reactive dialdehyde and represents an alternative route of LDL oxidation,38,39 while carbonyl groups (aldehydes and ketones) produced on protein side chains after oxidation represent an established marker of protein oxidation.40 In our study, we observed significantly increased MDA and PCs levels in atherosclerotic carotid plaques compared with normal lesions, but there were no significant differences between the clinical samples stratified according to the clinicopathologic parameters employed. Our data suggest that the lipid peroxidation process, by increased MDA plaque production and protein oxidation, probably contribute to plaque progres-
712 Sigala et al
sion, mainly at early stages, but it seems that they are not associated with latter stages related to plaque vulnerability and disruption. In conclusion, our findings support that oxidative stress is an important modulator of atherosclerosis. Specifically, our observations point at ox-LDL and peroxynitrite as important markers associated with advanced stages of this disease, concerning plaque destabilization and rupture. These findings could be exploited in the future in the form of potential markers that could be used to assist in prevention and therapeutic decision. AUTHOR CONTRIBUTIONS Conception and design: FS, AK, VG, IA Analysis and interpretation: AK, PS, KF, VG, IA Data collection: FS, KF Writing the article: FS, AK Critical revision of the article: FS, AK, EI, VG, IA Final approval of the article: FS, AK, PS, KF, SM, EI, VG, IA Statistical analysis: SM, EI Obtained funding: FS, AK, VG, IA Overall responsibility: VG FS and AK contributed equally to this work. REFERENCES 1. Yusuf S, Reddy S, Onupuus S, Anand S. Global burden of cardiovascular diseases: Part 1: General considerations, the epidemiologic transition, risk factors and impact of urbanization. Circulation 2001;104:2746-53. 2. Sacco RL, Benjamin EJ, Broderick JP, Dyken M, Easton JD, Feinberg WM, et al. American Heart Association Prevention Conference. IV. Prevention and Rehabilitation of Stroke. Risk factors. Stroke 1997;28: 1507-17. 3. North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. N Engl J Med 1991;325:445-53. 4. Aikawa M, Libby P. The vulnerable atherosclerotic plaque: pathogenesis and therapeutic approach. Cardiovasc Pathol 2004;1:125-38. 5. Koenig W, Khusevinova N. Biomarkers of atherosclerotic plaque instability and rupture. Arterioscler Thromb Biol 2007;27:15-26. 6. Lamon BD, Haijar DP. Inflammation at the molecular interface of atherogenesis: an anthropological journey. Am J Pathol 2008;173: 1253-64. 7. Sigala F, Georgopoulos S, Papalambros E, Chasiotis D, Vourliotaki G, Niforou A, et al. Heregulin, cysteine rich-61 and matrix metalloproteinase 9 expression in human carotid atherosclerotic plaques: relationship with clinical data. Eur J Vasc Endovasc Surg 2006;32:238-5. 8. Papalambros E, Sigala F, Georgopoulos S, Panou N, Kavantzas N, Agapitos M, et al. Vascular endothelial growth factor and matrix metalloproteinase-9 expression in human carotid atherosclerotic plaques: relationship with plaque destabilization via neovascularization. Cerebrovasc Dis 2004;18:160-5. 9. Schulze PC, Lee RT. Oxidative stress and atherosclerosis. Curr Atheroscler Rep 2005;7:242-8. 10. Parthasarathy S, Steinberg D, Witztum JL. The role of oxidized lowdensity lipoproteins in the pathogenesis of atherosclerosis. Annu Rev Med 1992;43:219-25. 11. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 1997;272:20963-6. 12. Napoli C, De Nigris F, Palinski W. Multiple role of reactive oxygen species in the arterial wall. J Cell Biochem 2001;82:674-82. 13. Matsunaga T. Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins. J Biochem 2001;130:285-93.
JOURNAL OF VASCULAR SURGERY September 2010
14. Reiters CD, Teng RJ, Eckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 2000;270:32460-6. 15. Rajagopalan S, Meng XP, Ramasamy S, Harrison DG, Glis ZS. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. J Clin Invest 1996;98:2572-9. 16. Egi K, Conrad N, Kwan J, Schulze C, Schulz R, Wildhirt SM. Inhibition of the inducible nitric oxide synthase and superoxide production reduces matrix metalloproteinase-9 activity and restores coronary vasomotor function in rat cardiac allografts. Eur J Cardiothorac Surg 2004;26:262-9. 17. Mu H, Wang X, Lin P, Yao Q, Chen C. Nitrotyrosine promotes human aortic smooth muscle cell migration throughoxidative stress and ERK1/2 activation. Biochim Biophys Acta 2008;1783:1576-84. 18. Huang Y, Mironova M, Lopes-Virella MF. Oxidized LDL stimulates matrix metalloproteinase-1 expression in human vascular endothelial cells. Arterioscler Thromb Vasc Biol 1999;19:2640-7. 19. Xu XP, Meisel SR, Ong JM, Kaul S, Cercek B, Rajavashisth TB. Oxidized low-density lipoprotein regulates matrix metalloproteinase-9 and its tissue inhibitor in human monocyte-derived macrophages. Circulation 1999;99:993-8. 20. Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 1997;272:20313-6. 21. Hernandez E, Goel N, Dougherty KG, Strickman NE, Krajcer Z. Benefits of catheter thrombectomy during carotid stenting. Tex Heart Inst J 2009;36:404-8. 22. Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W Jr, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 1995;92:1355-74. 23. Gorgoulis VG, Pratsinis H, Zacharatos P, Demoliou C, Sigala F, Asimacopoulos PA, et al. p53-Dependent ICAM-1 overexpression in senescent human cells identified in atherosclerotic lesions. Lab Invest 2005;85:502-11. 24. Andreadou I, Iliodromitis EK, Tsovolas K, Aggeli IK, Zoga A, Gaitanaki C, et al. Acute administration of Vitamin E triggers preconditioning via KATP channels and cyclic-GMP without inhibiting lipid peroxidation. Free Radic Biol Med 2006;41:1092-9. 25. Andreadou I, Iliodromitis EK, Mikros E, Constantinou M, Agalias A, Magiatis P, et al. The olive constituent oleuropein exhibits antiischemic, anti-oxidative and hypolipidemic effects in anesthetized rabbits. J Nutr 2006;136:2213-9. 26. Loftus IM, Naylor AR, Bell PR, Thompson MM. Matrix metalloproteinases and atherosclerotic plaque instability. Br J Surg 2002;89: 680-94. 27. Stocker R, Keaney JF Jr. New insights on oxidative stress in the artery wall. J Thromb Haemost 2005;3:1825-34. 28. Nishi K, Itabe H, Uno M, Kitazato KT, Horiguchi H, Shinno K, Nagahiro S. Oxidized LDL in carotid plaques and plasma associates with plaque instability. Arterioscler Thromb Vasc Biol 2002;22: 1649-54. 29. Uno M, Kitazato KT, Suzue A, Itabe H, Hao L, Nagahiro S. Contribution of an imbalance between oxidant-antioxidant systems to plaque vulnerability in patients with carotid artery stenosis. J Neurosurg 2005; 103:518-22. 30. Mannheim D, Herrmann JH, Versari D, Gössl M, Meyer FB, McConnell JP, et al. Enhanced expression of LP-PLA2 and lysophosphatidylcholine in symptomatic carotid atherosclerotic plaques. Stroke 2008; 39:1448-55. 31. Quehenberger O. Molecular mechanisms regulating monocyte recruitment in atherosclerosis. J Lipid Res 2005;46:1582-90. 32. JialalI I. Evolving lipoprotein risk factors: lipoprotein(a) and oxidized low-density lipoprotein. Clin Chem 1998;44:1827-32. 33. Clinton SK, Libby P. Cytokines and growth factors in atherogenesis. Arch Path Lab Med 1992;116:1292-300. 34. Clinton I. Oxidized low density lipoprotein and atherosclerosis. Int J Clin Lab Res 1996;26:178-84.
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
Sigala et al 713
35. Leeuwenburhg C, Hardy MM, Hazen SL, Wagner P, Ohishi S, Steinbrecher UP, et al. Reactive nitrogen Intermediates promote low density lipoprotein Oxidation in human atherosclerotic intima. J Biol Chem 1997;272:1433-6. 36. Hunter GC, Henderson AM, Westerbamd A, Kobayashi H, Suzuki F, Yan Z, et al. The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque. J Vasc Surg 1999;30:36-50. 37. Loftus IM, Naylor AR, Goodall S, Crowther M, Jones L, Bell PR, Thompson MM. Increased matrix metalloproteinase activity in unstable carotid plaques. A potential role in acute plaque disruption. Stroke 2005;31:40-47.
38. Ross R. The pathogenesis of atherosclerosis. A perspective for the 1990s. Nature 1993;362:801-9. 39. Minuz P, Fava C, Lechi A. Lipid peroxidation, isoprostanes and vascular damage. Pharmacol Rep 2006;57:57-68. 40. Dalle-Done I, Rossi R, Giustarini D, Milzani A, Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 2003;329:23-38.
Submitted Dec 18, 2009; accepted Mar 20, 2010.
AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of Journal of Vascular Surgery for the preceding 5 years are maintained and are available for purchase from Mosby until inventory is depleted. Please write to Elsevier Inc., Subscription Customer Service, 11830 Westline Industrial Drive, St. Louis, MO, or call 800-654-2452 (U.S. and Canada); 314-453-7041 (outside U.S. and Canada) for information on availability of particular issues and prices.