Published online before print October 17, 2008, doi: 10.1161/ATVBAHA.108.173658
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© 2009 American Heart Association, Inc.
Clinical and Population Studies |
Carotid Atherosclerotic Plaques Stabilize After Stroke
Insights Into the Natural Process of Atherosclerotic Plaque Stabilization
From the Experimental Cardiology Laboratory (W.P., W.E.H., D.P.V.d.K., G.P.), the Department of Vascular Surgery (W.E.H., F.L.M.), and the Department of Pathology (A.V.), University Medical Centre Utrecht; Interuniversity Cardiology Institute of the Netherlands (W.P., D.P.V.d.K.); and the Department of Vascular Surgery (J.P.P.M.d.V.), St. Antonius Hospital, Nieuwegein, The Netherlands.
Correspondence to W. Peeters, MD, Experimental Cardiology Laboratory, Heidelberglaan 100, Room G02-523, PO Box 3584 CX Utrecht, The Netherlands. E-mail w.peeters-2{at}umcutrecht.nl
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Abstract |
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Objective— Rupture of unstable atherosclerotic plaques is the pathological substrate for acute ischemic events. Underlying cellular and molecular characteristics of plaque rupture have been studied extensively. However, the natural course of symptomatic plaque remodeling after ischemic events is relatively unexplored.
Methods and Results— Atherosclerotic carotid plaques were obtained from 804 symptomatic (stroke=204 and TIA=426) and asymptomatic (n=174) patients undergoing carotid endarterectomy. The presence of macrophages, smooth muscle cells (SMC), collagen, calcification, and lipid-core size were assessed histologically. At protein level, inflammatory mediators (interleukin [IL]-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, interferon-gamma [INF-
], tumor necrosis factor-alpha [TNF-
], matrix degrading proteinases (MMPs), and an apoptosis marker (caspase-3) were determined. We associated plaque characteristics with time elapsed between the latest event and surgery. Early after stroke and TIA, plaques revealed an unstable phenotype. After stroke, the content of macrophages decreased significantly with time (P=0.02), whereas SMC content tended to increase. At protein level, IL-6, IL-8 expression levels and caspase activity strongly decreased after stroke or TIA.
Conclusions— Symptomatic carotid lesions remodel into more stable plaques over time after stroke. Changes in IL-6 and IL-8 and caspase preceded the decrease of macrophages. These temporal phenotypic plaque alterations should be taken into account for biomarker and therapeutic target validation studies using human atherosclerotic plaques.
The composition of 804 atherosclerotic plaques from patients who underwent carotid endarterectomy has been related to the time, elapsed between the latest cerebrovascular ischemic event and surgical excision. After stroke as well after TIA, the inflammatory status of the plaque decreased independent from potential confounders at histological and protein level.
Key Words: atherosclerosis • carotid arteries • inflammation • plaque • stroke
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Introduction |
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Thrombosis of the vulnerable atherosclerotic plaque is the predominant pathological substrate of acute cerebrovascular and cardiovascular events such as stroke and myocardial infarction (MI). Characteristics of the so-called unstable atherosclerotic plaque, which are supposed to contribute to the initial event of plaque rupture, have been described extensively.1–5 Current concepts, describing the histological features of the unstable atherosclerotic plaque, mainly originate from cross-sectional post mortem studies. The natural history of plaque progression and destabilization is unknown, but it has been suggested that progression of atherosclerosis is a summation of sequential repetitive events resulting in plaque stabilization and plaque destabilization. Most research on plaque progression has been focused on the cellular and molecular structure of the plaque that may precede local rupture.2,4–6 The alterations in plaque phenotype after a clinical event are relatively unexplored. To identify the natural history of plaque remodeling after a thrombotic event, we examined the structure of carotid endarterectomy specimen at histological and protein level in relation to the time elapsed between the most recent cerebrovascular event and surgery. For this purpose we used plaque samples and medical data from the multi center study "Athero-Express," including symptomatic and asymptomatic patients who had undergone carotid endarterectomy. Besides histological characteristics we assessed mediators that have been associated with the unstable plaque like cytokines, matrix metalloproteinases (MMP) and an apoptosis marker in 804 protein samples.7–10 The search for therapeutic options to prevent plaque destabilization is hampered by the lack of surrogate markers of disease progression. Therapeutic and diagnostic molecular targets often require validation of expression levels in human atherosclerotic plaques. In this study we assessed to what extent the outcome of validation studies may be influenced by the time that elapsed between the clinical event and the dissection of the atherosclerotic plaque. We report that after stroke, plaques remodel into a noninflammatory stable phenotype. The outcome of this study supports the concept that plaque stabilization and destabilization are sequential events in the progression of atherosclerotic disease.
See accompanying article on page 3
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Materials and Methods |
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Patient Inclusion
Athero-Express is an ongoing longitudinal vascular biobank study with the main objective to study the predictive value of local plaque markers as determinants for future cardiovascular events.10 All patients undergoing carotid endarterectomy (CEA) in two participating Dutch hospitals are asked to participate in the study. The Medical Ethics Committee of both hospitals approved the study, and participants provided written informed consent.
For the current research questions we have studied the atherosclerotic plaques from 804 consecutive patients (symptomatic [n=630] and asymptomatic [n=174]) who were included between April 2002 and June 2007. The indication for CEA for asymptomatic patients was based on the recommendations published by the Asymptomatic Carotid Surgery Trail (ACST) and for symptomatic patients the indication was based on recommendations based on the European Carotid Surgery Trail (ECST) and the North American Symptomatic Carotid Endarterectomy Trail (NASCET).11–14
From all patients, baseline data were obtained by extensive questionnaires including history of vascular disease, cardiovascular risk factors, and medication use (supplemental Table I, available online at http://atvb.ahajournals.org). Symptoms were categorized as described in the online supplement. Results from asymptomatic patients served as control values in comparison with data obtained from patients suffering from TIA or stroke. All data are provided for stroke and TIA patients separately.
Time Scale
To assess changes in plaque characteristics at histological and protein levels (plaque remodeling) in relation to time after the clinical event, plaques were categorized by the time episode that elapsed between the latest event and the surgical intervention. Symptomatic patients were categorized into 4 different groups according to the delay between event and surgery as described in detail in the online supplement.
Plaque Processing
Directly after excision, the atherosclerotic plaque specimen was taken to the laboratory for processing. The atherosclerotic lesions were dissected into 5-mm segments. The segment having the greatest plaque area was defined as the culprit lesion and was processed to assess the plaque phenotype for histological analyses as described previously and in detail in the online supplements.10,15 Recently, we have demonstrated that the segments adjacent to the culprit lesion showed good correlations with histological characteristics, that the histological analyses were also well reproducible and revealed an acceptable inter observer agreement.16
For measuring changes in plaque composition at protein level, expression of proteins playing a role in inflammatory pathways or plaque destabilization, such as cytokines (IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, INF-, and TNF-), matrix metalloproteinases (MMP-2, MMP-8, and MMP-9) and apoptosis marker caspase-3, were determined in 568 plaques from symptomatic and asymptomatic patients (stroke, n=139; TIA, n=303; asymptomatic, n=126). For detailed descriptions about protein isolation we refer to the online supplements.
Statistical Analysis
For statistical analyses of baseline characteristics in relation to the time episodes, the Kruskall Wallis test and the 
2 test were used. Protein analyses were performed by the Mann-Whitney U test, Kruskall Wallis test, and Bivariate Spearman’s Correlation test. The categorized histological plaque characteristics were analyzed by the 2 test. Data analysis was performed by using the statistical software SPSS 15.0 (SPSS Inc). For detailed descriptions of statistical analyses we refer to the online supplements.
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Results |
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Baseline Characteristics
The time interval between the latest ipsilateral event and surgery was clearly documented for all patients. Baseline characteristics for the different groups are documented in supplemental Table I. Plaques from patients who suffered from a TIA represented the majority of symptomatic plaques (68%, n=426) compared with stroke (32%, n=204). No differences in cardiovascular risk factors or drug use between the time categories were observed (supplemental Table I). In addition, we included 174 plaques from asymptomatic patients, which served as control values.
Histological Plaque Characteristics Early (<30 days) After Stroke or TIA
Tables 1 and 2
show the histological plaque characteristics for stroke and TIA, in relation to the time after the ischemic events. In carotid plaques obtained from patients operated early after stroke, macrophage infiltration tended to be higher compared with asymptomatic patients (stroke 71.1% versus asymptomatic 59.8%; Figure 1A). Early after stroke (<30 days), the SMC content was significantly lower in comparison with the asymptomatic group (63.2% versus 78% P=0.03; Table 1, Figure 1B). Moreover, after stroke a significantly higher proportion of plaques revealed a large lipid core (39.5% versus 24.9%, P=0.03). These differences were not observed in the TIA group compared to the asymptomatic group.
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Protein Expression Levels and Activity Early (<30 Days) After Stroke or TIA
In comparison with asymptomatic patients, plaques from stroke patients demonstrated significantly elevated levels of proinflammatory cytokines IL-6 (132.4 [56.1 to 208.8] versus 82.9 [59.8 to 106.2], P=0.03) and IL-8 (838.5 [118.9 to 1557.9] versus 158.4 [63.5 to 253.3], P<0.001) when operated within 30 days after the event (Table II see www.ahajournals.org, Figure 1CD). In these plaques the increased levels of IL-6 positively correlated with the expression levels of IL-2 (P=0.009 R=.558) and TNF- (P=0.04 R=.447). IL-8 did not show correlations with other proinflammatory cytokines.
Activity of MMP-8 (10.1[4.9 to 15.3] versus 4.4 [3.6 to 5.1], P=0.005), MMP-9 (0.92 [0.66 to 1.18] versus 0.72 [0.58 to 0.87], P=0.04), and caspase (12844 [8257 to 17 430] versus 8566 [7392 to 9739], P=0.02) were also significantly elevated in carotid lesions harvested from patients who had suffered from a recent (<30 days) stroke (supplemental Table II, Figure 1E, supplemental Figure IIBC).
Within 30 days after TIA, IL-8 levels (301.3 [179.4 to 423.2] versus 158.4 [63.5 to 253.3], P<0.001) and MMP-8 activity (6.4 [5.0 to 7.9] versus 4.4 [3.6 to 5.1], P=0.004) were significantly increased compared to plaques from asymptomatic patients (supplemental Table III, Figure 1D, supplemental Figure IIB). IL-6 expression levels were higher compared to plaques from asymptomatic patients and correlated positively with IL-2 (P=0.007 R=.297), IL-12p70 (P=0.01, R=.273), INF- (P=0.04, R=.220), TNF- (P=0.002 R=.337).
Histological Plaque Characteristics in Relation to Time After Stroke or TIA
The proportion of plaques demonstrating moderate/heavy macrophage staining after stroke decreased nearly 2-fold over time (71.1%–36.8%, P=0.02) and was below the control value after 180 days (asymptomatic patients 59.8%; Figure 1A). Macrophage decrease sustained over time in plaques from stroke patients who used statins preoperatively (82.1% to 33.3%, P=0.03; Table 1). Ninety days after stroke, the proportion of plaques with moderate/heavy SMC staining increased significantly (P=0.01) to the control values. These observations suggest that unstable atherosclerotic plaques remodel and stabilize over time after stroke (Figure 1B).
After TIA, no decrease in macrophage content was observed over time. In the TIA group the proportion of plaques containing macrophages tended to increase after more than 180 days (52.3% versus 77.1%, P=0.07) (Figure 1A). The increase was also evident in the group of patients who used statins preoperatively (52.5% versus 81.5%; Table 2). The proportion of plaques containing macrophages also tended to increase after more than 180 days in this group (52.5% versus 81.5%, P=0.02). Furthermore, in the TIA group collagen content reduced over time (84.4% versus 72.2%, P=0.04; Table 2).
Protein Expression Levels and Activity in Relation to Time After Stroke or TIA
After stroke, IL-6 levels strongly decreased within 90 to 180 days (132.4 [56.1 to 208.8] versus 49.4 [34.3 to 64.5], P=0.02) (supplemental Table II, Figure 1C). IL-8 levels demonstrated an early decrease and a ten fold reduction at later time points (>180 days; 838.5 [118.9 to 1557.9] versus 75.4 [25.9 to 124.8], P<0.001) reaching levels compared to control values of asymptomatic patients (supplemental Table II, Figure 1D). Caspase activity reduced significantly (12844 [8257 to 17430] versus 8366 [5357 to 11374], P=0.04; Figure 1E) over 180 days after stroke, and resulted in nearly similar activity in comparison with asymptomatic patients (8566 [7392 to 9739]). Surprisingly, MMP activity did not show significant alterations over time, although MMP-8 activity revealed a trend toward reduction within the first 180 days after stroke (10.1 [4.9 to 15.3] versus 6.3 [4.3 to 8.3], P=0.06).
In plaques from patients who suffered from TIA, IL-8 levels decreased over 180 days after TIA (301.3 [70.1 to 198.6] versus 163.5 [61.7 to 265.3], P=0.03; supplemental Table III, Figure 1D). Other IL levels and MMP- or caspase activity barely demonstrated consistent changes after the event. However, temporal fluctuations were observed in IL expression levels (IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, INF-, and TNF-) in the TIA group. These changes were not consistent over time, but elevated levels were mainly observed 90 to 180 days after the event (supplemental Table III).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Progression of atherosclerosis is a process with repetitive events including cellular and molecular alterations leading to plaque stabilization or plaque destabilization, which may eventually result in acute cerebrovascular and cardiovascular events. The natural history of atherosclerotic plaque stabilization and destabilization is not completely understood. We studied the temporal sequence of carotid plaque characteristics at histological and protein level after ischemic cerebrovascular events.
In this study, we demonstrate that the atherosclerotic carotid plaques from symptomatic patients reveal an unstable phenotype early after the acute ischemic event, which is in line with previous observations.1,3,17 To determine the natural history of plaque progression after a cerebrovascular event, the plaque phenotype has been associated with different time episodes between the latest ischemic event and surgery. After stroke, plaques reveal a significant reduction of macrophage content in relation to time and a clear trend of increasing numbers of smooth muscle cells. Plaques from patients who suffered from a TIA did not demonstrate pathohistological changes with increasing time intervals. These findings support previous observations from Redgrave and colleagues, who also suggested significantly reduced macrophage infiltration in relation to time after stroke.18 In the aforementioned study of Redgrave et al conclusions regarding the first time episode were drawn on a limited number of plaques. However, this study comprised a large number of samples, which were equally distributed over the different episodes resulting in a sufficiently powered study.
In the current study we also determined plaque protein expressions as a fingerprint of the inflammatory status in relation to time after an event. We showed that IL-6 and IL-8 levels and caspase activity were significantly increased in lesions harvested early after the initial event compared to asymptomatic lesions, which indicates that symptomatic lesions demonstrate an increased vulnerable plaque phenotype. These cytokine expression levels and caspase activity altered subsequently over time in atherosclerotic carotid plaques after stroke or TIA. IL-6 and IL-8 are expressed in atherosclerotic tissue and have proinflammatory properties by activating and recruiting inflammatory cells and stimulating matrix degrading enzymes, which contribute to plaque instability and lead to cardiovascular events.19–24 Lower IL levels were observed early after stroke, suggesting that plaque composition alters very early after the initial event toward a more stable plaque phenotype. The remarkable increase of cytokine levels after more than 90 days after TIA may point to a different pathophysiology compared to stroke and reflects the clinical recurrent pattern of cerebrovascular events after 90 days as described by Johnston et al.25
Apoptosis of macrophages and smooth muscle cells is another prominent feature in atherosclerotic tissue and is associated with plaque destabilization.7,9,26–28 Macrophages contribute to the majority of apoptotic cells within the plaque.27 Caspase-3 can be considered as an apoptosis marker because it plays a key role in the apoptotic pathways.29 The results of this study indicate that the activity of the execution caspase-3 strongly reduced after stroke, which also points to alterations toward a more stable plaque phenotype.
The mechanisms for plaque stabilization after acute stroke or TIA remain to be investigated. The observed phenotypic changes may reflect a normal response to injury, where inflammation decrease precedes smooth muscle cell migration, matrix deposition, and tissue remodeling. However, in conditions of high oxidative stress like atherosclerosis, nitric oxide (NO) and peroxynitrite induce apoptotic cell death. Apoptosis in atherosclerotic plaques is mainly present in advanced atherosclerotic tissue containing numerous foam cells and macrophages.30,31 Macrophages contribute to the majority of apoptotic cells and express high levels of inducible NO synthase (iNOS) and nitrotyrosine as a footprint of peroxynitrite in hypoxic conditions. Under these circumstances, NO targets DNA and induces oxidative damage resulting in oxidative modification and deamination of DNA.32 DNA damage may lead subsequently to apoptotic cell death and decreased macrophage concentrations. The decrease in caspase activity after stroke may be a result from decreased apoptosis over time.
Besides inflammation markers and mediators of apoptosis, the current study has examined the course of MMP activity after ischemic events within the atherosclerotic plaque. Matrix metalloproteinases are associated with degradation of the extracellular matrix and thereby with plaque destabilization.8 Macrophages in the atherosclerotic plaque are a major source of matrix metalloproteinases. We assessed the MMP-2, MMP-8, and MMP-9 activity in the atherosclerotic plaque in relation to time. Although macrophage infiltration decreased over time, MMP-levels did not change after stroke. A reasonable explanation is that MMPs can also be produced by other cell types like SMCs, which tended to increase with time after stroke, thereby balancing the macrophage related effects on MMP levels. Furthermore, it has been suggested that MMPs are associated with SMC migration suggesting that gelatinases play a role in lesion stabilization.33
Patient characteristics and drug use were associated with the different time intervals to determine possible confounding effects. No differences between the groups have been observed, indicating that the possible confounders such as preoperative drug use could not explain the results.
The pleiotropic immune-modulatory properties of statins have gained much attention in the last decade.34 Besides their primary lipid lowering effect, statins may exert antiinflammatory effects in cardiovascular patients. Additional analyses with respect to patients who used statins preoperatively in the stroke and TIA group demonstrated that the decrease of macrophage infiltration over time was still present.
It has already been described that biomarkers, such as plasma-derived IL-6 and IL-8, have predictive value for future cardiovascular events.21,22,35,36 Researchers and pharmaceutical companies use human atherosclerotic plaques to validate expression levels of proteins of interest. Carotid plaques are of specific interest because these are plaques originating from patent vessels and regularly obtained in patients who are still alive. The Athero-Express study has been initiated to examine local plaque biomarkers that are predictive for local and systemic progression of atherosclerotic disease. Recently, we have shown that local inflammatory lipid rich plaques are associated with lower restenosis rates after endarterectomy.15 The current study indicates that inflammatory proteins (IL-6 and IL-8) are associated with clinical presentation and reveals expression patterns that rapidly change over time in atherosclerotic plaques after an event. Insight in temporal changes in protein expression, in plaques from patients who suffered from stroke or TIA, is relevant for therapeutic and diagnostic research. For instance, levels of diagnostic biomarkers for prediction of secondary manifestations of cardiovascular disease should ideally remain stable over time after an acute event.
Limitations of the Current Study
Our observations may have been influenced by cardiovascular risk factors or drug use. Patients in the >180 days-group were included in the first phase of the Athero-Express study. We have looked at variations in drug use and risk factors over time and did not observe differences between patient groups. Therefore we may conclude that drug use is not a confounding factor.
The current study examined protein levels in a lower number of atherosclerotic plaques compared with histology. However, the selection of patients for protein expression has been unbiased. Therefore, we feel that this large sample can be considered representative of the total cohort. Histological stainings of inflammatory cytokines IL-6 and IL-8 were not successful. We were not able to correlate macrophage presence with cytokine expression within the atherosclerotic plaque. Therefore, we cannot make inferences regarding the association between the decrease of cytokine expression and the decreased macrophage presence. The retrospective aspect of this study weakens the strength of our observations. Ideally prospective studies would be needed to reproduce these observations, for instance by monitoring the unstable plaque with MRI or CT-Pet fusion techniques. Nowadays, it would be complicated to execute such a study, because symptomatic patients are being operated much faster after the event.37
Conclusion
This study demonstrates that the delay between the latest onset of stroke and surgery is significantly associated with the macrophage content of the plaque and a decrease of IL-6 and IL-8 levels and caspase activity. These observations suggest that plaque stabilization and destabilization are sequential events in atherosclerotic disease progression. Carotid plaques are frequently used for drug and biomarker target validation. Based on these observations we suggest that these validation studies merit careful consideration if the temporal aspect of plaque remodeling has not been taken into account.
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Acknowledgments |
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Sources of funding
The reported work was supported by the European Union (grant EU OIF21773).
Disclosures
None.
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Footnotes |
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W.P. and W.E.H. contributed equally to this study.
Original received April 9, 2008; final version accepted September 19, 2008.
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References |
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