Advanced Carotid Plaques Research Articles

Antioxidant Status and Purine Bases in Carotid Artery Plaque

Free radical excess and oxidative stress are implicated in the formation and progression of atherosclerotic plaque through actions on susceptible vascular cells, such as by activating xanthine oxidase. Purine bases and other antioxidant compounds could play important protective roles in atherogenesis, as could nonenzymatic low molecular weight thiol defenses, not previously evaluated in carotid artery plaque. Therefore, we measured purine catabolites (hypoxanthine, xanthine, uric acid, allantoin) and antioxidant compounds (total sulphydryl groups, homocysteine, cysteine, and glutathione) in advanced carotid artery plaque and found a high ratio of allantoin to uric acid, suggesting a ongoing local oxidative stress.

Dynamin and perforin are associated with neovascularisation in advanced carotid plaques

Intimal plaque neovascularization is associated with the development of symptomatic disease and thrombosis, with new 'leaky' fragile microvessels prone to haemorrhage. Perforin or pore forming protein is involved in vascular cell death by forming pores in target cells. Enzymes, in particular, granzyme B are secreted by immune infiltrates present in inflammatory plaque regions and have been shown to induce endothelial cell apoptosis. Similarly, dynamin-2 is a GTPase which mediates oxidised low density lipoprotein-induced apoptosis and is also required for granzyme B-mediated exocytosis and apoptosis. Our pilot studies identified increased expression of these proteins in complicated atherosclerotic plaques. Here we demonstrate by immunohistochemistry that both proteins are over-expressed in angiogenic regions of complicated carotid plaques. Dynamin-2 was extensively localised around microvessels and in immune infiltrating cells whilst perforin was localised in immune infiltrating cells, endothelial cells and smooth muscle cells. Over-expression of these proteins may contribute to plaque destabilisation by increasing cellular apoptosis in vulnerable atherosclerotic plaques.

Endothelial Apoptosis Does Not Determine Symptom Status in Carotid Artery Disease

Background: We examined the hypothesis that endothelial denudation in advanced carotid plaques (CPs) occurs by increased apoptosis of endothelial cells (ECs) using scanning electron microscopy (SEM) as well as markers of cellular proliferation and apoptosis in advanced symptomatic CPs (SCPs) and asymptomatic CPs (ACPs). Methods: 93 consecutive patients underwent carotid endarterectomy. Five additional specimens were studied by SEM. We performed TUNEL assays, and immunostaining against Fas receptor (FasR), Fas ligand (FasL), activated caspase 3 (ACA3) and Ki-67. Results: SEM revealed morphological changes consistent with EC detachment. Surprisingly, ACA3 positivity was more pronounced on the endothelium of ACPs (4.6 ± 0.7% of total EC count) than on SCPs (3.3 ± 0.7%, p = 0.049), and was found to correlate positively with nuclear Ki-67 expression (r_s = 0.275, p = 0.040). FasL expression was significantly increased on the endothelium of SCPs compared with ACPs (66.4 ± 4.4 vs. 53.9 ± 4.5%, p = 0.047). Conclusions: Absence of increased positivity of apoptotic markers dismisses apoptosis as a dominant mechanism underlying endothelial detachment of SCPs. Rather, increased ACA3 with co-expression of Ki-67 in ACPs might suggest that renewal of endothelium by active cell turnover may contribute to clinically silent evolution of plaques with preserved EC integrity. These observations may assist in designing novel therapies to prevent endothelial decay and symptom generation in advanced carotid artery disease.

Purine Catabolism in Advanced Carotid Artery Plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.

Endogenous Expression of C-Reactive Protein Is Increased in Active (Ulcerated Noncomplicated) Human Carotid Artery Plaques

There is growing evidence suggesting that C-reactive protein (CRP) is an effecter molecule able to induce and promote atherothrombosis. The presence of CRP in atherosclerotic plaques may reflect local production or infiltration from circulating CRP increased in general inflammatory responses. Our aim was to analyze the presence of CRP in human advanced carotid artery plaques with differential anatomo-pathological characteristics and to assess local expression of CRP and other proinflammatory genes in these lesions. Human carotid artery specimens from 38 patients undergoing scheduled endarterectomy were classified into 3 groups: ulcerated (noncomplicated) (UNC, n=19), fibrous (F, n=12) and ulcerated (complicated/hemorrhagic) plaques (UC, n=7). The presence of CRP was evaluated by immunohistochemistry, and plasma samples were screened for circulating high-sensitivity C-reactive protein. TaqMan Low-density Arrays were used for study of genes related to inflammation (CRP, interleukin-6, macrophage colony-stimulating factor-1, monocyte chemotactic protein-1, cyclooxygenase-2). CRP mRNA levels were predominantly detected in UNC-high risk plaques but not in UC (P=0.001). UNC also exhibit the highest expression levels of other genes involved in the inflammatory responses: cyclooxygenase-2 (P<0.005 versus F and versus UC), IL-6 (P<0.005 versus F and versus UC) and monocyte chemoattractant protein-1 (P<0.01 versus F and versus UC). Plaque CRP mRNA levels correlated with immunohistochemical findings but were independent of plasma high-sensitivity CRP. In UNC plaques endothelial cells and inflammatory cells were strongly positive for CRP around areas of newly formed microvessels. In human high-risk carotid artery plaques (UNC) CRP expression reflects an active proinflammatory stage. Local synthesis of CRP could be involved in plaque neovascularization and increased risk of hemorrhagic transformation.

In vivo accuracy of multisequence MR imaging for identifying unstable fibrous caps in advanced human carotid plaques.

To evaluate the in vivo accuracy of a multisequence MRI technique for prospectively identifying one feature of the vulnerable plaque-an unstable fibrous cap-in human carotid atherosclerosis. The carotid arteries of 18 endarterectomy patients were preoperatively imaged in a 1.5 T scanner using a multisequence protocol that generated four contrast weightings (3D time of flight (ToF), T1, proton density (PD), and T2) at each slice location. With the use of previously published MR criteria, the images of the vessel wall were first examined for evidence of an unstable fibrous cap. The imaging findings were then correlated with the histology from the surgical specimens. A blinded review of the MR findings with the histologic state of the fibrous cap revealed that 1). assessing the preoperative appearance of the fibrous cap has a high test sensitivity (0.81) and specificity (0.90) for identifying an unstable cap in vivo; and 2). the availability of different contrast weightings facilitated image interpretation when intimal calcifications or flow artifacts obscured the lumen surface. Multisequence MRI can accurately characterize the in vivo state of the fibrous cap. This finding supports the use of these noninvasive techniques to prospectively identify vulnerable plaques.

In vivo accuracy of multipectral magnetic resonance imaging for identifying lipid-rich necrotic cores and intraplaque hemorrhage in advanced human carotid plaques

In vivo accuracy of multipectral magnetic resonance imaging for identifying lipid-rich necrotic cores and intraplaque hemorrhage in advanced human carotid plaques

Elevated C-reactive protein constitutes an independent predictor of advanced carotid plaques in dyslipidemic subjects.

Inflammation plays a key role in the physiopathology of atherosclerosis. C-reactive protein (CRP) has been found to predict cardiac events in healthy subjects and in patients with coronary heart disease. However, the relationship between CRP and subclinical atherosclerosis is not well established. We examined the potential relationship between CRP and common carotid artery intima-media thickness and carotid plaques in dyslipidemic subjects. Dyslipidemic patients (n=1051) were recruited for the study. All patients had a complete clinical examination and systematically underwent ultrasonographic evaluation of the extracranial carotid arteries on a duplex system. The serum concentration of CRP was measured by using a sensitive immunoradiometric assay. In a univariate model, a strong positive relationship was found between CRP and the severity of carotid stenosis (P<0.0001). In multivariate analysis, the association between CRP and the degree of carotid atherosclerosis remained significant for advanced plaques (P=0.0007) in male subjects only. Significant correlations were found between CRP and body mass index (P<0.0001) and between CRP and other markers associated with the metabolic syndrome. In this large dyslipidemic population, elevated CRP is an independent predictor of advanced carotid plaques in male subjects. Body mass index and other markers of the metabolic syndrome (HDL cholesterol, triglycerides, diabetes, and high blood pressure) are significant determinants of CRP levels in this population.

Coexpression of endothelin-converting enzyme-1 and endothelin-1 in different stages of human atherosclerosis.

Endothelin-converting enzyme (ECE)-1 activates endothelin-1 (ET-1) and may thus contribute to the regulation of vascular tone and cell growth during atherosclerosis. To evaluate ECE-1 immunoreactivity concerning big ET-1/ET-1, we performed qualitative and quantitative immunohistochemistry in normal internal mammary arteries (n=10), in coronary arteries with adaptive intimal fibrosis (n=10), in aortic fatty streaks (n=10), and in distinct regions of advanced carotid plaques (n=15). Furthermore, we determined ECE-1 activity in the control specimens and in the inflammatory intimal regions of carotid plaques. Double immunolabeling showed that ECE-1 was present in endothelial cells, vascular smooth muscle cells, and macrophages. All ET-1(+) cells were simultaneously ECE-1(+). Most importantly, there were significantly more ET-1(+) cells in the intima and media when atherosclerosis was in an inflammatory stage than when it was in a noninflammatory stage. Moreover, ECE-1 activity was upregulated in the intima of carotid plaques, although immunohistochemically, there were no significant differences between the number of ECE(+) cells in the different compartments of the arterial wall. Together with ET-1, ECE-1 is abundantly present in human arteries and at different stages of atherosclerotic plaque evolution. The upregulation of the ECE-1/ET-1 system is closely linked to the presence of chronic inflammation and is present in very early stages of plaque evolution. Therefore, enhanced production of active ET-1 may substantially contribute to cell growth and the regulation of vascular tone in advanced atherosclerotic lesions and in the very early stages of plaque evolution, when a plaque is still imperceptible clinically.

Carotid plaque typing by multiple-parameter ultrasonic tissue characterization

We evaluated the ability of ultrasonic tissue characterization (UTC), based on backscattered echo signals, to distinguish among the components of advanced carotid plaques. We performed spectral analysis of echo signals acquired from human carotid endarterectomy specimens in vitro to calculate three parameters of the calibrated power spectrum: slope, intercept and total power for fibrous, lipid pool and thrombus constituents of plaque. Plaque constituents were identified histologically. We evaluated classification efficacy by discriminant function analysis. Slope and intercept parameters alone provided correct classification in 92.5%, 57.6% and 72.4% of fibrous, lipid pool and thrombus plaque components, respectively. Slope, intercept and total power used in combination improved classification of the three tissue types to 93.0%, 69.7% and 81.0%. The overall proportion of correctly classified tissue regions increased from 84.5% to 88.0% by the combined use of the three parameters. The improvement in classification that occurred when we included total power as a third parameter suggests that ultrasound plaque components may not consist solely of small, randomly distributed isotropic scatterers. Our ability to identify plaque thrombi provides motivation for future studies of parameter-based imaging methods for identifying such plaque that presents an increased risk of embolic neurologic ischemic events.

Diversity of T-cell antigen receptor V beta gene utilization in advanced human atheroma.

Human atheromata contain T lymphocytes, but knowledge of the function and receptor specificity of these cells is limited. Immunohistochemical studies have established that T cells in advanced human carotid plaques express predominantly the alpha/beta form of the T-cell receptor (TCR). We then compared the use of variable region genes of the beta-chain (V beta) of the TCR for antigen by analysis of 14 carotid plaques and peripheral blood samples obtained at carotid endarterectomy. We used a direct approach that avoids isolation and culture of T cells. RNA extracted from lesions and peripheral blood mononuclear cells was reverse transcribed and amplified by polymerase chain reaction (PCR) to determine rearrangements of 18 V beta sequences. PCR products were visualized on Southern blots using a probe internal to the PCR primers. Input cDNA from lesions and peripheral blood was adjusted to yield equivalent signals for a conserved region of the TCR beta-chain to permit comparisons. As expected, utilization of TCR V beta genes in peripheral blood cells was nonselective: an average of 17 of 18 V beta regions yielded signals (n = 14). Frequency of variable-region gene usage in lesions and blood was highly concordant: of 252 sequences tested (14 samples, 18 sequences per sample), 240 were identified in peripheral blood versus 207 in plaques. V beta genes 10 and 11 were not expressed in plaques, a significant difference when compared with peripheral blood (P = .0001 by chi 2). However, the remaining 16 genes showed no significant differences. This analysis indicates that T cells generally express a diverse pattern of V beta genes within complex human atheroma.