Carotid Plaque Pathology
Thrombosis, Ulceration, and Stroke Pathogenesis
Background and Purpose— To determine the relationship between ulceration, thrombus, and calcification of carotid artery atherosclerotic plaques and symptoms of ipsilateral or contralateral stroke.
Methods— We compared microscopic plaque morphology from patients with and without stroke symptoms ipsilateral or contralateral to the plaque. Plaques were characterized for ulceration, thrombus, and calcification. We analyzed plaques from 241 subjects: 170 patients enrolled in the Asymptomatic Carotid Atherosclerosis Study (ACAS) and 71 patients enrolled in the North American Symptomatic Carotid Endarterectomy Trial (NASCET); 128 subjects had no history of stroke symptoms, 80 subjects had ipsilateral symptoms, and 33 had contralateral symptoms.
Results— Plaque ulceration was more common in plaques taken from symptomatic patients than those without symptoms (36% versus 14%; P<0.001); frequency of ulceration was similar for plaques associated with ipsilateral (34%) and contralateral (42%) symptoms. Thrombus was most common in plaques taken from patients with both ipsilateral symptoms and ulceration. The extent of calcification was unassociated with stroke symptoms.
Conclusion— Carotid plaque ulceration and thrombosis are more prevalent in symptomatic patients. Ulceration is more common in symptomatic patients regardless of side of carotid symptoms, whereas thrombus is associated with ipsilateral symptoms and plaque ulceration. Preoperative identification of carotid ulceration and thrombus should lead to greater efficacy of stroke prevention by carotid endarterectomy.
Carotid artery disease is a well-established risk factor for ischemic stroke. Surgical removal of carotid plaques reduces risk of stroke in symptomatic and asymptomatic individuals.1,2 Nevertheless, the relationship between carotid plaque morphology and stroke pathogenesis is poorly understood.
Two large prospective randomized trials have provided important information regarding the role of carotid endarterectomy.1,2 The North American Symptomatic Carotid Endarterectomy Trial (NASCET) and the Asymptomatic Carotid Atherosclerosis Study (ACAS) have shown clinical effectiveness of endarterectomy in substantial groups of patients with carotid artery disease. NASCET has shown benefit for carotid artery surgery in patients with symptomatic disease of 50% or more stenosis; ACAS has shown benefit for surgery for asymptomatic disease of 60% or more stenosis in a selected population of patients if the surgical morbidity/mortality is <3% and the patient has at least a 5-year prognosis for a healthy life thereafter.
For the coronary vasculature, extensive work has described progression from the early raised lesion to the later complicated plaque.3 Calcification, ulceration, and thrombosis are the major pathologic events in the transformation of the early asymptomatic lesion to the mature plaque producing myocardial ischemia.3 The role of these pathologic events in the development of brain ischemia is uncertain.
See Editorial Comment, pg 257
Carotid plaques removed from patients undergoing carotid endarterectomy represent a potentially important source of information explaining the roles of critical pathologic events in stroke pathogenesis. Park showed that grossly visible carotid plaque ulceration is more prevalent in patients who have a history of stroke or transient ischemic attack (TIA).4 Rothwell demonstrated that radiographic evidence of carotid plaque surface irregularity in symptomatic arteries was associated with surface irregularity in contralateral arteries.5 We hypothesized that important differences exist between symptomatic and asymptomatic plaques. In this study, we compare the prevalence of ulceration, thrombosis, and calcification in carotid plaques taken from symptomatic and asymptomatic individuals.
Materials and Methods
Carotid artery atherosclerotic plaques were surgically removed from patients enrolled in ACAS and NASCET. Participating NASCET and ACAS centers sent formalin-fixed carotid artery plaque specimens from patients to the central pathology laboratory at University of Southern California. These plaques were then x-rayed, photographed, and processed; plaques excessively fragmented (>2 pieces) were not subject to further analysis. Microscopic sections were then taken at 3-mm intervals over the 2-cm region of maximum plaque pathology.
Using an eyepiece micrometer, one observer characterized microscopic sections for presence of ulceration and thrombosis. Microscopic sections were analyzed with the observer blinded to the clinical status of the patients whose plaques were being examined. Ulceration was considered present when a depression below, or disruption of, the plaque surface of 560 mm or more resulted in exposure of substantial amount of lipid (either cholesterol crystals or lipid-laden macrophages) to lumen surface (Figure 1). Thrombus was considered present when the lumen surface was covered by thrombotic material larger than 340×110 μm. In preliminary studies, use of these dimensions (by eyepiece micrometer) was found to identify a substantial proportion of atherosclerotic plaques that had presence of ulceration or thrombus.6 Kappa values calculated for these variables (ulceration and thrombus) were 0.77 and 0.93.
We x-rayed the formalin-fixed plaques to determine the extent of plaque calcification. These images were analyzed by computer using a Quantimet 970 Image Analysis System and the Quips software package (Cambridge Instruments Ltd, United Kingdom). X-ray negatives of plaques were digitized using a video camera and stored as a 512×512 pixel matrix. The image was displayed as a combination of a gray image and a binary overlay representing the detected region. An amend routine was used to measure total plaque area and area of calcification after calibrating against images of known dimension.
Subjects were classified by history of stroke or TIA. We characterized patients as: asymptomatic (ie, lacking ischemic symptoms); ipsilateral symptomatic (ie, symptomatic with history of symptoms ipsilateral to side of endarterectomy); and contralateral symptomatic (ie, symptomatic with history of symptoms contralateral to side of endarterectomy). Percent stenosis was determined by preoperative arteriogram using NASCET criteria. Differences between groups were tested using χ2 tests (for categorical variables) and t tests (for continuous variables). Two-sided P<0.05 was considered significant.
We analyzed carotid artery plaques from 241 subjects, 170 from ACAS and 71 from NASCET. The mean age of subjects was 67 years (range, 48 to 80 years) and 65% were male. There were 128 asymptomatic subjects, 80 ipsilateral symptomatic patients, and 33 contralateral symptomatic patients. For the 113 subjects with symptoms, the median interval between event and endarterectomy was 48 days. Percent stenoses data were available for 230 subjects: median stenoses for asymptomatic, ipsilateral symptomatic, and contralateral symptomatic patients were 75%, 64%, and 67%.
Ulceration was more prevalent in plaques taken from patients with symptoms (ipsilateral or contralateral) than those from asymptomatic patients (Table; 36% versus 14%; P<0.001). Frequency of ulceration was similar for plaques taken from patients with ipsilateral or with contralateral symptoms (34% versus 42%; Figure 2).
Thrombus was only slightly more prevalent in plaques taken from ipsilateral or contralateral symptomatic patients (23%) compared with asymptomatic subjects (18%), and thrombus prevalence did not differ between patients with ipsilateral versus contralateral symptoms (24% versus 21%). However, among patients with ipsilateral symptoms, presence of thrombus and presence of ulcer were strongly associated (P<0.005; Figure 3)
There was no significant association between presence of thrombus or ulceration and interval between symptoms and surgery: subjects with symptoms ≤30 days before endarterectomy had prevalence of ulceration and thrombus of 41% (17/41) and 29% (12/41); for those subjects with symptoms >30 days before endarterectomy, the frequency of ulceration and thrombus was 33% (24/72) and 19% (14/72). Presence of thrombus or ulceration was also unrelated to percent stenosis: presence of ulceration or thrombus for patients with <60% stenosis, 60% to 69% stenosis, 70% to 79% stenosis, or ≥80% stenosis was 39% (19/49), 37% (23/63), 35% (19/55), and 36% (27/74). Median percent calcification, determined for 196 plaques, was similar for plaques taken from asymptomatic (16%), ipsilateral symptomatic (15%), and contralateral symptomatic patients (15%).
Among the 71 NASCET subjects, there was no significant association between ulceration and: (1) hemisphere versus retinal event (P=0.48); (2) lacunar versus nonlacunar event (P=0.72); (3) stroke versus TIA (P=0.89); or (4) presence or absence of ipsilateral infarct on computed tomography (P=0.44). Among the NASCET patients, there was no significant association between thrombus and hemisphere versus retinal event (P=0.84) and presence or absence of ipsilateral infarct on computed tomography (P=0.73). Thrombus was present in 43% (6/14) of patients with lacunar events versus 18% (7/40) of patients with nonlacunar events (P=0.06). Thrombus was also present in 35% (11/32) of patients with stroke versus 18% (7/39) or patients with TIA (P=0.11).
We show that carotid plaque ulceration is more prevalent in plaques taken from symptomatic patients. Ulceration prevalence is comparable for both the ipsilateral symptomatic and contralateral symptomatic patient groups. Thrombus, in contrast, is most common in plaques with both ipsilateral symptoms and ulceration. These findings provide important new insights into the pathogenesis of ischemic symptoms from carotid artery atherosclerotic disease.
Plaque ulceration is a complex process that requires chronic increase in shear stress at sites of stenosis and sudden changes in intra-arterial pressure.3 Both intraplaque inflammation and metalloproteinase activity are initiators of ulceration.7,8 Plaque erosions, both superficial and deep, are invariably associated with activated macrophages and T lymphocytes.9,10 Plaque macrophages tend to localize to high-shear regions of plaques, in contrast to smooth muscle cells that are more prominent in low-shear regions. Other manifestations of inflammation, including expression of inflammatory cytokines, are more common in symptomatic carotid plaques.11,12 Demonstrations of the Gram-negative organism Chlamydia pneumoniae in atherosclerotic lesions, including carotid plaques,13,14 suggest a linkage between systemic infection and focal atherosclerotic inflammatory changes.
Metalloproteinases within plaques are also linked to plaque ulceration.8 Mast cell-derived proteases appear to have an important role in the activation of metalloproteinases, resulting in degradation of plaque wall and subsequent ulceration.8 Mast cells, along with smooth muscle cells and macrophages, also appear to have a role in plaque calcification.15 The current study found no relationship between the extent of plaque calcification and symptoms, consistent with previous work.16
Atherosclerotic plaques have a predisposition to thrombosis. Previous work has demonstrated a profound procoagulant milieu within plaques. Plasminogen activator inhibitor-1, the primary inhibitor of tissue plasminogen activator, is overexpressed within plaques, creating a net antifibrinolytic effect.17 Tissue factor, the primary generator of the coagulation cascade, is expressed by critical elements of atherosclerotic lesions.18 The extent of tissue factor activity in plaques is partially dependent on the extent and distribution of tissue factor pathway inhibition within plaques.19 Tissue factor within plaques is localized to areas of extensive lipid deposition, particularly lipid-laden macrophages (foam cells) and cholesterol crystals.20 Note that our definition of microscopic ulceration requires presence of significant amount of lipid on the lumen surface. Therefore, plaque ulceration, as observed in this study, represents an important site for thrombus formation on the plaque surface.
Based on findings presented herein, as well as previous observations, we suggest that patients destined to be symptomatic from carotid artery disease are more likely to first have plaque ulceration, which tends to occur irrespective of what ultimately will be the side of brain ischemia.5 Ulceration thus represents an important substrate from which thrombus and symptoms may later arise. Thrombus, superimposed on ulceration, appears to be important for production of brain ischemia. The ischemic episode itself may be produced by microembolism or by hemodynamic insufficiency.
We show that carotid plaque ulceration and thrombus are associated with different subsets of “symptomatic” patients. Ulceration is more common in patients with symptoms either ipsilateral or contralateral to the plaque; thrombus is more common in patients with both ipsilateral symptoms and plaque ulceration. These 2 patient subsets are important in helping to define distinctions between “symptomatic patients” and “symptomatic plaques.” This distinction has typically depended on presence (or absence) of symptoms ipsilateral to the lesion. A carotid artery atherosclerotic plaque, in a patient with only contralateral symptoms of brain ischemia, may sometimes be considered “asymptomatic”; a patient with such a lesion may be grouped with patients who are completely asymptomatic. Our data demonstrate important pathological differences between plaques from patients with and without symptoms, regardless of symptom laterality. Therefore, for future trials of therapy of carotid artery disease, it may be appropriate to consider carotid plaques with contralateral symptoms as “symptomatic.”
Our study is limited by a number of potentially important confounding factors. We evaluated surgical specimens that all had longitudinal incisions, thus creating the potential for artifact. This latter possibility was limited, but not eliminated, by the blinded analysis of nonfragmented plaques. Also note that only a limited number of ACAS and NASCET centers participated in this study, and plaques were typically not sent in consecutive series; for example, excessively fragmented specimens were usually not submitted for analysis. Moreover, these plaques were often not removed from patients chronologically near the time of symptoms. This interval between symptoms and surgery may have contributed to the presence of plaque ulceration or thrombus in a minority of our plaques. Note, however, that our criteria for presence of ulceration or thrombus required a substantial-sized lesion; a morphometric requirement of smaller lesion dimensions may have resulted in a higher proportion of plaques deemed abnormal. Finally, our analysis did not include intraplaque hemorrhage; the latter has recently been described as an important contributor to atherogenesis and plaque destabilitzation.21
In conclusion, carotid plaque ulceration and thrombosis are more prevalent in symptomatic patients. Ulceration is more common in symptomatic patients regardless of side of carotid symptoms, whereas thrombus is associated with ipsilateral symptoms and plaque ulceration. Preoperative identification of carotid ulceration and thrombus by either ultrasound or arteriography is currently quite limited,22,23 and improvements in the relevant technologies should lead to greater efficacy of stroke prevention by carotid endarterectomy.
Participating ACAS Centers
Albuquerque VA Medical Center, New Mexico; University of Arizona Health Sciences Center; Bowman Gray School of Medicine, Winston-Salem, North Carolina; Columbia Presbyterian Medical Center, New York; East Orange Medical Center, New Jersey; Francis Scott Key Medical Center, Baltimore, Maryland; Harbin Clinic, Rome, Georgia; Henry Ford Hospital, Detroit, Michigan; Hershey Medical Center, Pennsylvania; University of Kentucky, Lexington, Kentucky; Hospital de L’Enfant-Jesus, Quebec; Little Rock VA Medical Center, Arkansas; Loyola University Medical Center, Maywood, Illinois; Marshfield Medical Clinic, Marshfield, Wisconsin; New England Medical Center, Boston, Massachusetts; Ochsner Clinic, New Orleans, Louisiana; Oregon Health Sciences University School of Medicine, Portland, Oregon; Richmond VA Medical Center, Richmond, Virginia; Saint John’s Mercy Medical Center, St. Louis, Missouri; San Diego VA Medical Center, San Diego, California; Singing River Hospital, Pascagoula, Mississippi; Sunnybrook Health Science Center, Toronto, Ontario; UCLA Medical Center; Virginia Mason Research Center, Seattle, Washington. A full list of collaborators from these centers is provided in reference 2.
Participating NASCET Centers
University of Illinois Hospital Chicago, Illinois (C. Helgason); Northwestern University Chicago, Illinois (B. Cohen); Ohio State University Columbus, Ohio (A. Slivka) Dartmouth-Hitchcock Medical Center Lebanon, New Hampshire (A. Reeves); University of Texas Health Sciences (J. Grotta); UCLA School of Medicine, California (W. Moore); USC School of Medicine Los Angeles, California (M. Fisher); Madison VAMC Madison, Wisconsin (C. Acher); Marshfield Medical Clinic Marshfield, Wisconsin (P. Karanjia); Mississauga Hospital, Mississauga, Ontario (G. Sawa); Hospital de L’Enfant Jesus Quebec City, PQ (D. Simard); Richmond Eye & Ear Hospital Richmond, Virginia (J. Harbison); VA Medical Center Syracuse, New York (A. Culebras); Sunnybrook Health Science Center Toronto, Ontario (J. Norris); Vancouver General Hospital UBC Health Sciences Center Vancouver, BC (P. Teal).
This work was supported by National Institutes Health NS20989, NS06655, and NS24456. We thank Paul S. Reehal, MD for his assistance in preparing this manuscript.
- Received November 6, 2003.
- Revision received August 26, 2004.
- Accepted September 21, 2004.
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Yamashita K, Ouchi K, Shirai M, Gondo T, Nakagawa T, Ito H. Distribution of Chlamydia pneumoniae infection in the atherosclerotic carotid artery. Stroke. 1998; 29: 773–778.
Raghunath PN, Tomaszewski JE, Brady ST, Caron RJ, Okada SS, Barnathan ES. Plasminogen activator system in human coronary atherosclerosis. Arterioscler Thromb Vasc Biol. 1995; 15: 1432–1443.
Caplice NM, Mueske CS, Kleppe LS, Simari RD. Presence of tissue factor pathway inhibitor in human atherosclerotic plaques is associated with reduced tissue factor activity. Circulation. 1998; 98: 1051–1057.
Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A. 1989; 86: 2839–2843.
Streifler JY, Eliasziw M, Fox AJ, Benavente OR, Hachinski VC, Ferguson GG, Barnett HJ. Angiographic detection of carotid plaque ulceration. Comparison with surgical observations in a multicenter study. North American Symptomatic Carotid Endarterectomy Trial. Stroke. 1994; 25: 1130–1132.