Cap Inflammation Research Articles

PS-C28-6: HIGH TESTOSTERONE AND LOW ESTRADIOL SERA LEVELS ARE ASSOCIATED WITH UNSTABLE PLAQUE COMPOSITION IN MEN WITH SEVERE CAROTID ATHEROSCLEROSIS

Background and Objectives: Ischemic strokes caused by the rupture of unstable atherosclerotic plaques in the carotid arteries, are leading causes of mortality and disability worldwide. Sex differences exist in plaque composition, where men are more likely to develop unstable plaques than women. Sex hormones affect vascular reactivity and inflammation, but it remains unclear if they are involved in plaque instability and how their role differs in men and women. Herein, we investigated the association between circulating endogenous sex hormone levels and plaque composition/instability in men and women with severe carotid atherosclerosis. Design and method: Pre-operative fasting blood samples and plaque specimens were collected from post-menopausal women and men greater than 55 years old with severe carotid atherosclerosis who underwent a carotid endarterectomy at McGill-affiliated hospitals in Montreal, Canada. Plaque composition and stability were examined by experienced vascular pathologists according to gold-standard histological classifications. Liquid chromatography-tandem mass spectrometry-based methods were developed to quantify endogenous sex hormones, 17ß-estradiol (E2), testosterone, androstenedione, and dehydroepiandrosterone, in sera samples. Results: Subjects (n = 460) had a mean age of 71.0 ± 9.0, were 68.9% male, and had various comorbidities (hypercholesterolemia [82.6%], hypertension [84.6%], type 2 diabetes [34.5%]). Clinical characteristics were similar among post-menopausal women (n = 143) and men (n = 317) with stable versus unstable plaques. Men had a significantly greater proportion of unstable versus stable plaques compared to women (men unstable: 61.5%, women unstable: 41.3%; P < 0.001), characterized predominantly by a large lipid-rich core, hemorrhage, and high presence of inflammatory cell infiltration. Men with unstable plaques had significantly higher levels of testosterone than men with stable plaques (P = 0.004). In men, testosterone levels were inversely associated with fibrosis (P < 0.05), while directly associated with unstable plaque features, including greater lipid core size (P < 0.05), and a greater presence of foam cells (P < 0.05), neovascularization (P < 0.01), and plaque (P < 0.01) and cap inflammation (P < 0.05). No associations were observed between testosterone levels and plaque features in women. Endogenous E2 levels were inversely associated with neovascularization (P < 0.05) and plaque and cap inflammation (P < 0.001) in men, while inversely associated with fibrosis levels in women (P < 0.05). Conclusion: Our findings suggest that higher testosterone and lower E2 levels are associated with unstable plaque composition in men with carotid atherosclerotic disease and may partly explain the higher proportion of unstable plaques observed among men than women of similar age and comorbidity status. Instead, in women, estradiol appears to play a detrimental role in plaque instability by inversely affecting fibrous tissue levels (a key stabilizing feature of plaques).

Cap inflammation leads to higher plaque cap strain and lower cap stress: An MRI-PET/CT-based FSI modeling approach

Plaque rupture may be triggered by extreme stress/strain conditions. Inflammation is also implicated and can be imaged using novel imaging techniques. The impact of cap inflammation on plaque stress/strain and flow shear stress were investigated. A patient-specific MRI-PET/CT-based modeling approach was used to develop 3D fluid-structure interaction models and investigate the impact of inflammation on plaque stress/strain conditions for better plaque assessment. 18FDG-PET/CT and MRI data were acquired from 4 male patients (average age: 66) to assess plaque characteristics and inflammation. Material stiffness for the fibrous cap was adjusted lower to reflect cap weakening causing by inflammation. Setting stiffness ratio (SR) to be 1.0 (fibrous tissue) for baseline, results for SR=0.5, 0.25, and 0.1 were obtained. Thin cap and hypertension were also considered. Combining results from the 4 patients, mean cap stress from 729 cap nodes was lowered by 25.2% as SR went from 1.0 to 0.1. Mean cap strain value for SR=0.1 was 0.313, 114% higher than that from SR=1.0 model. The thin cap SR=0.1 model had 40% mean cap stress decrease and 81% cap strain increase compared with SR=1.0 model. The hypertension SR=0.1 model had 19.5% cap stress decrease and 98.6% cap strain increase compared with SR=1.0 model. Differences of flow shear stress with 4 different SR values were limited (<10%). Cap inflammation may lead to large cap strain conditions when combined with thin cap and hypertension. Inflammation also led to lower cap stress. This shows the influence of inflammation on stress/strain calculations which are closely related to plaque assessment.

Abstract 588: Cap Inflammation Leads to Large Plaque Cap Stress Decrease and Strain Increase: MRI-PET/CT-Based FSI Modeling

Introduction: Recent studies indicated that plaque rupture may be associated with: a) extreme mechanical stress/strain conditions; b) large lipid-rich necrotic core; c) thin and weakening fibrous cap; d) cap inflammation; e) intraplaque hemorrhage. Fayad et al and others have been developing multi-modality imaging technology using PET/CT (Positron Emission Tomography/ Computed Tomography) and MRI (magnetic resonance imaging) to identify inflammation in arteries. The goal of this paper is to investigate possible impact of cap inflammation on plaque stress/strain and flow shear stress conditions. Method: An MRI-PET/CT-based modeling approach is proposed to develop fluid-structure interaction (FSI) models for human carotid plaque assessment and quantify the effect of inflammation on plaque stress/strain conditions. The combined PET/CT and MRI data was acquired from a 74 year old male patient (with informed consent) to assess arterial inflammation. The patient was imaged with dark blood multi-contrast MRI and 18F-fluorodeoxyglucose (18F-FDG) PET/CT in separate imaging sessions 12 days apart. For the 15-slice MRI/PET/CT data set, inflammation was identified for S4-S11. The 3D FSI model was built following established procedures. Material stiffness for the fibrous cap was adjusted lower to reflect the cap weakening causing by cap inflammation. Setting stiffness ratio (SR) to be 1.0 for baseline, results for SR=0.5, 0.25, and 0.1 (smaller SR value indicates softer cap stiffness) were obtained. Results: Mean plaque cap stress (PC-Stress), strain (PC-Strain) and flow shear stress (PC-FSS) are reported in Table 1. Roughly, 30-50% decrease in PC-Stress and 50-100% increase in PC-Strain were observed. Greater changes were observed for SR=0.1. Conclusion: Since cap inflammation and extreme stress/strain conditions are of great interest, further investigations are warranted to validate our findings.

Relationship between Carotid Artery Remodeling and Plaque Vulnerability with T1-Weighted Magnetic Resonance Imaging

The aim of this study was to validate the relationship between carotid artery remodeling defined as the carotid remodeling index (CRI) and plaque vulnerability by comparing the degree of outward remodeling calculated using 3-dimensional inversion recovery-based T1-weighted imaging (magnetization-prepared rapid acquisition gradient echo [MPRAGE]) with the symptomatology and histology of plaques extracted during carotid endarterectomy. Sixty-one patients with 50% stenosis or more (North American Symptomatic Carotid Endarterectomy Trial criteria) were included. The average rate of stenosis was 79.8%. The CRI was determined by measuring the external cross-sectional vessel area (CSVA) at the maximum stenosis of the internal carotid artery (ICA) and dividing it by the external CSVA at the distal ICA (unaffected by atherosclerosis) using MPRAGE imaging. The CRI was significantly higher in symptomatic patients compared with asymptomatic patients (1.98±.26 versus 1.68±.24, P<.0001). A higher CRI positively correlated with the necrotic core area (r=.57, P<.0001) and negatively correlated with the fibrous cap thickness (r=-.33, P=.01). It was also significantly associated with severe intraplaque hemorrhage (P<.0001) and the prevalence of cap inflammation with macrophage (P=.03) and lymphocyte (P=.01) infiltration. The larger outward remodeling of the carotid artery on MPRAGE imaging had symptomatic carotid plaques and histologically vulnerable plaques. This study indicates that MPRAGE imaging is useful for the assessment of carotid artery remodeling.

Coronary calcification identifies the vulnerable patient rather than the vulnerable Plaque

ObjectivePresence of coronary artery calcium (CAC) is associated with a high risk of adverse cardiovascular outcomes. Nevertheless, although CAC is a marker of atherosclerosis it is still uncertain whether CAC is a marker of plaque vulnerability. Therefore, the aim of this study was to verify if calcification identifies a vulnerable patient rather than the vulnerable plaque. MethodsA morphologic and morphometric study on 960 coronary segments (CS) of 2 groups of patients was performed: (i) 17 patients who died from AMI (510 CS); (ii) 15 age-matched control patients without cardiac history (CTRL, 450 CS). ResultsCalcification was found in 47% CS of AMI and in 24.5% CS of CTRL. The area of calcification was significantly higher in AMI compared to CTRL (p = 0.001). An inverse correlation was found between the extension of calcification and cap inflammation (r2 = 0.017; p = 0.003). Multivariate regression analysis demonstrated that the calcification was not correlated with the presence of unstable plaques (p = 0.65). Similarly, the distance of calcification from the lumen did not represent an instability factor (p = 0.68). ConclusionThe present study suggests that CAC score evaluation represents a valid method to define the generic risk of acute coronary events in a population, but it is not useful to identify the vulnerable plaque that need to be treated in order to prevent an acute event.

Abstract WP162: Impact of Carotid Artery Remodeling by T1 weighted MR Imaging for the Assessment of Plaque Vulnerability in High-grade Carotid Stenosis

Background- The relationship between coronary artery remodeling and plaque vulnerability has been described on the basis of symptomatology and histology. However, the association with carotid artery remodeling has not been explored in detail. The aim of this study was to validate the relationship between carotid artery remodeling and plaque vulnerability by comparing the degree of outward remodeling calculated using 3D inversion-recovery-based T1-weighted imaging (magnetization-prepared rapid acquisition gradient-echo [MPRAGE]) with the symptomatology and histology of plaques extracted during carotid endarterectomy (CEA). Methods and Results- Sixty-one patients with high-grade carotid stenosis who underwent CEA and whose plaque could be examined were included. The average rate of stenosis as per the NASCET criteria was 79.8%. The carotid remodeling index (CRI) was determined by measuring the external cross-sectional vessel area (CSVA) of the maximum stenosis of the internal carotid artery (ICA) and dividing it by the external CSVA of the distal ICA unaffected by atherosclerosis using MPRAGE imaging. The relationship between the CRI and plaque vulnerability was evaluated on the basis of symptomatology and histology. The CRI was significantly higher in symptomatic patients than in asymptomatic patients (1.98 ± 0.26 vs. 1.68 ± 0.24, p &lt; 0.0001). A higher CRI was positively correlated with the necrotic core area (r = 0.568, p &lt; 0.0001) as well as significantly associated with severe intraplaque hemorrhage (p &lt; 0.0001) and the prevalence of cap inflammation with macrophage (p = 0.03) and lymphocyte (p = 0.01) infiltration. Conclusion- These results validate the relationship between carotid artery remodeling and plaque vulnerability in high-grade carotid stenosis. MPRAGE imaging is effective to assess plaque vulnerability in terms of the CRI in addition to the signal intensity of carotid plaques.

Features of Unstable Carotid Plaque During and After the Hyperacute Period Following TIA/Stroke

The aim was to test the hypothesis that histologically unstable carotid plaque features were more prevalent in patients undergoing carotid endarterectomy (CEA) in the acute period after onset of symptoms and that the plaque would assume more stable histological characteristics as the delay from the most recent event increased. Seven histological features of plaque instability (haemorrhage, large lipid core, chronic plaque inflammation, chronic cap inflammation, marked vascularity, cap rupture and many foam cells) were independently quantified and then correlated with recency of symptoms in patients undergoing CEA. In patients undergoing CEA ≤14 days of their last event, 87/119 (73%) exhibited ≥5/7 unstable histological plaque features, compared with 22/40 (55%) of patients undergoing delayed surgery (p = .048). As expected, there was a sustained decline in the prevalence of unstable plaque features in 61 patients undergoing surgery between days 7-28. However, there was then a marked increase in the prevalence of plaque haemorrhage (59% up to 65%), large lipid core (41% up to 78%), chronic plaque inflammation (71% up to 91%), cap rupture (35% up to 39%), many foam cells (24% up to 43%) and marked vascularity (71% up to 91%) in 23 patients undergoing CEA after 29 days had elapsed. Patients undergoing surgery ≤14 days had a significantly higher overall burden of high risk plaque features compared with those undergoing delayed CEA. However, the secondary upsurge across a range of unstable plaque features in patients undergoing CEA after ≥29 days had elapsed suggests that the relationship between recency of symptoms and plaque histology is more complex than had been anticipated in previous studies.

Features of Unstable Carotid Plaque During and After the Hyperacute Period Following TIA/Stroke

BackgroundThe aim was to test the hypothesis that histologically unstable carotid plaque features were more prevalent in patients undergoing carotid endarterectomy (CEA) in the acute period after onset of symptoms and that the plaque would assume more stable histological characteristics as the delay from the most recent event increased. MethodsSeven histological features of plaque instability (haemorrhage, large lipid core, chronic plaque inflammation, chronic cap inflammation, marked vascularity, cap rupture and many foam cells) were independently quantified and then correlated with recency of symptoms in patients undergoing CEA. ResultsIn patients undergoing CEA ≤14 days of their last event, 87/119 (73%) exhibited ≥5/7 unstable histological plaque features, compared with 22/40 (55%) of patients undergoing delayed surgery (P = 0.048). As expected, there was a sustained decline in the prevalence of unstable plaque features in 61 patients undergoing surgery between days 7–28. However, there was then a marked increase in the prevalence of plaque haemorrhage (59% up to 65%), large lipid core (41% up to 78%), chronic plaque inflammation (71% up to 91%), cap rupture (35% up to 39%), many foam cells (24% up to 43%) and marked vascularity (71% up to 91%) in 23 patients undergoing CEA after 29 days had elapsed. ConclusionPatients undergoing surgery ≤14 days had a significantly higher overall burden of high risk plaque features compared with those undergoing delayed CEA. However, the secondary upsurge across a range of unstable plaque features in patients undergoing CEA after ≥29 days had elapsed suggests that the relationship between recency of symptoms and plaque histology is more complex than had been anticipated in previous studies.

A robust rabbit model of human atherosclerosis and atherothrombosis

Disruption and thrombosis of atherosclerotic plaques cause most acute cardiovascular events, but their systematic study has been hampered by the lack of suitable animal models. To assess the value of a modified rabbit model of atherothrombosis, we performed detailed histology of rabbit aortic plaques. Atherosclerosis was induced with a high cholesterol diet fed 2 weeks prior to and 6 weeks after balloon injury of the aorta, followed by 4 weeks of normal diet. We found six out of eight types of plaques cataloged by the American Heart Association in the rabbit aorta. Vulnerable plaques were defined as those with attached platelet and fibrin-rich thrombi after pharmacological triggering with Russell's viper venom and histamine. Ruptured plaques had, as also described for human plaques: i) marked medial and adventitial changes, including neovascularization and inflammation; ii) cholesterol monohydrate crystals and liquid crystalline cholesterol esters in the intima and the fibrous cap; and iii) inflamed, thin fibrous caps. Increased cholesterol monohydrate area, internal elastic lamina area, positive remodeling, fibrous cap inflammation, adventitia breakdown, and inflammation were independent predictors of plaque disruption. Our findings reveal novel insights into plaque vulnerability and could guide the design of noninvasive imaging approaches for detecting and treating high-risk plaques.

Clinical Feasibility of Molecular Imaging of Plaque Inflammation in Atherosclerosis

Despite substantial advances in the diagnosis and management of coronary artery disease, acute coronary events continue to occur in many patients. It has been increasingly realized that the lesions responsible for acute events may not necessarily be critically obstructive and hence not be associated with inducible ischemia. Various morphologic features of plaque vulnerability have been described by CT angiography, intravascular ultrasound, and optical coherence tomography. The culprit plaques often demonstrate large plaque and necrotic core volumes, positive vascular remodeling, and attenuation of fibrous plaque caps. The remaining obligatory component of plaque vulnerability is fibrous cap inflammation; molecular imaging is best suited for identification of monocyte-macrophage infiltration. Whereas multiple candidate targets have been evaluated in preclinical molecular imaging studies, only (18)F-FDG and (99m)Tc-annexin-A5 have been recently used in the settings of acute vascular events. These 2 imaging strategies have demonstrated the clinical feasibility of imaging for detection of inflammation.

Human Carotid Plaque Calcification and Vulnerability

Background: Inflammation is a key mechanism in human atherosclerotic plaque vulnerability and disruption. The objective was to determine the differential gene expression of pro- and anti-inflammatory factors in the fibrous cap and shoulder region of noncalcified and calcified carotid endarterectomy plaques. Methods: Thirty carotid endarterectomy plaques were classified as type Va (noncalcified, n = 15) and type Vb (calcified, n = 15) in accordance with the American Heart Association consensus. Using laser capture microdissection, fibrous cap and shoulder regions were excisedfrom frozen sections. Gene expression of pro- [interleukin 1 (IL-1), IL-8 and monocyte chemoattractant protein 1 (MCP-1)] and anti-inflammatory (IL-10) factors, and bone formation (bone morphogenetic protein 6 and osteocalcin) mediators were quantitated by real-time PCR. Protein levels were determined using Western blotting. Results: Mean percent carotid stenosis and calcification area were 79 and 5% in Va-plaques (40% symptomatic) and 77 and 42% in Vb-plaques (20% symptomatic). Macrophages infiltrating the region of the fibrous cap and the shoulder were more numerous in non-calcified plaques compared with calcified plaques (p < 0.01]. mRNA expression of MCP-1 and IL-8, and protein levels of IL-8 were also greater in Va plaques compared to Vb plaques (p < 0.05). Protein levels and mRNA expression of osteocalcin were greater in Vb compared to Va plaques (p < 0.05). Conclusions: Fibrous cap inflammation is more likely to occur in noncalcified than in calcified plaques. These findings suggest that carotid atherosclerotic plaque calcification is a structural marker of plaque stability.

Calcification and carotid plaque vulnerability:inverse relationship between the degree of calcification and fibrous cap pro-inflammatory gene expression

Objective Inflammation and calcification are common phenomenons in human atherosclerotic plaque.The objective of this study was to determine the gene expression of some proteins and anti-inflammatory factors as well as bone formation mediators in the fibrous cap and shoulder region of non-calcified and calcified carotid endarterectomy(CEA) plaques.Methods Twenty-two CEA plaques were classified as Va(non-calcified,n=11) and Vb(calcified,n=11) in accordance with the AHA consensus in 1995.Mean percent in carotid stenosis and calcification area was 76.4% and 0% in Va and 74.2% and 33.6% in Vb respectively as determined by quantitative histomorphometry.Using laser capture microdissection,the fibrous cap and shoulder regions were excised from 22 frozen sections/plaques.After total RNA extraction and reverse transcription,gene expression of proteins(IL-1,IL-8,and MCP-1),anti-inflammatory factor(IL-10) and bone formation protein(BMP-6 and Osteocalcin) were detected by Real-time PCR.Results mRNA expression of MCP-1,IL-8 and IL-1 in Va plaques were 1.8 fold higher than that in Vb plaques,mRNA expression of anti-inflammatory factor IL-10 and osteogenic related genes BMP-6 and Osteocalcin were 3.8,4.2,and 6.9 fold higher in Vb plaques compared to Va plaques.Conclusions Our findings indicate that fibrous cap inflammation and susceptibility to disruption is more likely to occur in non-calcified plaques. Key words: Arteriosclerosis/PP; Calcinosis; Chemokines; Gene expression

Degree of carotid plaque calcification in relation to symptomatic outcome and plaque inflammation

Objective We undertook this study to quantitate differences in the degree of calcification between symptomatic and asymptomatic plaques removed at carotid endarterectomy (CEA) and to determine associated extent of plaque macrophage infiltration, a histopathologic feature of plaque instability. Methods CEA plaques (n = 48) were imaged at 1.25-mm intervals with spiral computed tomography (CT; 10-15 images per plaque). Indications for CEA were transient ischemic attack (n = 16), stroke (n = 5), amaurosis (n = 4), and critical asymptomatic stenosis (n = 23). The percent area calcification for each plaque was determined in spiral CT serial sections and averaged for each plaque. In 31 of 48 plaques macrophage infiltration was quantitated in corresponding histologic sections with immunohistochemical techniques. Results The mean (± SD) age of patients with symptomatic and asymptomatic plaques was 66 ± 7 years vs 71 ± 7 years, respectively, and degree of stenosis was 76% versus 82%, respectively ( P = .05). Atherosclerosis risk factors were similar between groups. Percent plaque area calcification was twofold greater in asymptomatic versus symptomatic plaques (48% ± 19% vs 24% ± 20%, respectively; P < .05). At receiver operating characteristic curve analysis, 80% of symptomatic plaques were below and 87% of asymptomatic plaques were above a cutoff point of 30% plaque area calcification. Macrophage burden was greater in the symptomatic plaques than in the asymptomatic plaques (52% vs 23%; P < .03). A strong inverse relationship between the degree of plaque calcification and macrophage infiltration was found in critical carotid stenoses ( r = −0.87; P < .001). Conclusions Symptomatic plaques are less calcified and more inflamed than asymptomatic plaques. Regardless of clinical outcome, a strong inverse correlation was found between the extent of carotid plaque calcification and the intensity of plaque fibrous cap inflammation as determined by the degree of macrophage infiltration. Carotid plaque calcification is associated with plaque stability, and is a potential spiral CT in vivo quantitative marker for cerebrovascular ischemic event risk.

Apolipoprotein (a) fragments in relation to human carotid plaque instability

Purpose: An elevated plasma level of lipoprotein (a) is an independent risk factor for atherothrombotic cardiovascular disease by yet undefined mechanisms. We have previously reported that matrix metalloproteinases cleave apolipoprotein (a) into 2 main fragments, F1 and F2, the latter (the C-terminal domain) exhibiting in vitro a high-affinity binding to extracellular matrix components, including fibrin(ogen). We therefore tested the hypothesis that the lipoprotein (a) matrix metalloproteinase–derived F2 is localized in potentially or morphologically unstable human carotid plaque at regions of increased matrix metalloproteinase activity. Methods: Carotid plaques removed after endarterectomy (n = 18) were evaluated for structural features indicative of instability (thin fibrous cap, inflammation, and proximity of the necrotic core to the lumen); each plaque was classified as unstable (n = 10) or stable (n = 8). Western blot analysis was performed to quantitate apolipoprotein (a) and its fragments F1 and F2 in plaque extracts. Immunohistochemical staining was used to localize apolipoprotein (a) and its fragments within the atherosclerotic plaque. In situ zymography was used to determine regions of gelatinase (matrix metalloproteinase 2 and matrix metalloproteinase 9) activity. Results: Western blot analyses demonstrated a 2.5-fold higher density of F2 in unstable plaques than in stable plaques (3.07 ± 1.9 vs 1.18 ± 0.8; P <.05). In morphologically unstable plaques, there was preferential distribution of F2 within regions of fibrous cap inflammation and/or foam cell accumulation and within abluminal necrotic cores. In morphologically stable plaques, however, localization was predominantly found in the medial smooth muscle cells. Regions of enhanced matrix metalloproteinase 2 and matrix metalloproteinase 9 activity co-localized with the transmural distribution of F2 within the plaque. Conclusions: These findings suggest that F2 in regions of increased matrix metalloproteinase activity is a potential mechanism for superimposed thrombotic events in morphologically unstable human carotid plaques. The relationship between plasma lipoprotein (a) levels and accumulation of F2 and the potential correlation of F2 to human plaque disruption and thrombosis warrant further study. (J Vasc Surg 2000;32:555-63.)