Atherosclerotic Plaque Components Research Articles

Defining and Defending Murine Models of Plaque Rupture

There is general agreement that atherosclerotic plaque rupture is a major clinical problem and that finding ways to prevent it would be greatly facilitated by an animal model of plaque rupture. The article by Schwartz et al1 seeks to clarify and define the terms used in descriptions of disrupted atherosclerotic plaques, and then to apply these to murine models in order to assess their similarities to and differences from human ruptured lesions. On the face of it this seems a reasonable thing to do, but it has some serious conceptual problems. The first is the issue of whether it is useful at all to provide definitions of disrupted plaques, especially when these are based on histopathology. The second concerns the transferability of such definitions from human to mouse. I would like also to deal with issues relating to reevaluation of existing mouse models of plaque rupture. What do we gain from framing scientific definitions of real-world phenomena? In the context of the ruptured plaque, Schwartz et al1 argue that this would provide an agreed upon and consistent framework that would make it easier for different labs to compare and contrast their data. There is obviously virtue in this as a general principle in science, but we have to consider the possibility that in the developing field of plaque rupture it may be premature. We do not know why plaques rupture, we do not know whether “plaque erosions” are simply the adjacent manifestations of ruptures that are out of the plane of the section, and we do not know whether “plaque fissures” arise through a different set of processes to plaque ruptures. In other words, this means that we do not know whether ruptures, erosions and fissures are ontogenically similar or different. We are therefore moving beyond the boundaries …

MRI of atherosclerosis: diagnosis and monitoring therapy

Atherosclerosis is a prevalent disease affecting millions of Americans. Despite our advances in diagnosis and treatment, atherosclerosis is the leading cause of death in America. High-resolution magnetic resonance imaging has overcome the limitations of current angiographic techniques and has emerged as a leading noninvasive imaging modality for atherosclerotic disease. Atherosclerosis of the arterial wall of the human carotid, aortic, peripheral and coronary arteries have all been successfully evaluated. In addition, the power of magnetic resonance imaging to differentiate the major components of atherosclerotic plaque has been validated. The ability to image the vessel wall and risk stratify atherosclerotic plaque will create management decisions not previously faced, and has the potential to change the way atherosclerosis is treated.

Carotid plaque classification: Defining the certainty with which plaque components can be differentiated

Multicontrast-weighted MRI has the potential to become a powerful tool for assessment of atherosclerotic plaque. However, similarities in MR properties across plaque components limit the certainty with which these components can be differentiated. An understanding of MRI's underlying limitations in distinguishing atherosclerotic plaque components, and optimization of key parameters (including the set of components investigated and contrast weightings used) are required. In this study we analyzed endarterectomy specimens using multicontrast MRI and compared the results with matching histological findings to determine the probability of error, an unbiased measure of the underlying error caused by similarity in the spectral characteristics of components. The total error was >40% when five distinct components were investigated, but this was halved when components with similar functions and intensities were grouped together. When three contrast weightings were used to view plaque, diffusion-weighted imaging (DWI) proved valuable for separating hemorrhage from necrotic core, and "hemorrhage + necrotic" from "loose connective tissue + fibrous tissue." A two-way interaction between contrast weightings and components demonstrated that the value of a contrast can be exploited or marginalized depending on the choice of contrast weightings used.

Selective Clearance of Macrophages in Atherosclerotic Plaques by the Protein Synthesis Inhibitor Cycloheximide

Macrophages are an essential component of unstable atherosclerotic plaques and play a pivotal role in the destabilization process. We have demonstrated previously that local delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus selectively clears macrophages in rabbit plaques. Because mTOR controls mRNA translation, inhibition of protein synthesis might induce selective macrophage cell death. We therefore investigated in the present study the effect of the protein synthesis inhibitor cycloheximide on macrophage and smooth muscle cell (SMC) viability. In vitro studies with cultured macrophages and SMCs showed that cycloheximide induced selective apoptosis of macrophages in a concentration- and time-dependent manner. Moreover, macrophages could be selectively depleted in rabbit carotid artery rings with collar-induced atherosclerotic plaques after in vitro treatment with cycloheximide. Local in vivo administration of cycloheximide via osmotic minipumps to rabbit carotid arteries with collar-induced atherosclerotic plaques significantly reduced the macrophage but not the SMC content. Cycloheximide-treated plaques showed signs of apoptosis (increased terminal deoxynucleotidyl transferase end labeling and fluorescein isothiocyanate-Val-Ala-dl-Asp(O-methyl)-fluoromethylketone labeling) that did not colocalize with SMCs. Organ chamber studies demonstrated that the functionality of SMCs and the endothelium were not influenced by cycloheximide treatment. All together, these findings demonstrate that cycloheximide decreases the macrophage load in atherosclerotic plaques by induction of apoptosis without changing SMC content or contractility.

Automated classification of atherosclerotic plaque from magnetic resonance images using predictive models

The information contained within multicontrast magnetic resonance images (MRI) promises to improve tissue classification accuracy, once appropriately analyzed. Predictive models capture relationships empirically, from known outcomes thereby combining pattern classification with experience. In this study, we examine the applicability of predictive modeling for atherosclerotic plaque component classification of multicontrast ex vivo MR images using stained, histopathological sections as ground truth. Ten multicontrast images from seven human coronary artery specimens were obtained on a 9.4 T imaging system using multicontrast-weighted fast spin-echo (T1-, proton density-, and T2-weighted) imaging with 39-μm isotropic voxel size. Following initial data transformations, predictive modeling focused on automating the identification of specimen's plaque, lipid, and media. The outputs of these three models were used to calculate statistics such as total plaque burden and the ratio of hard plaque (fibrous tissue) to lipid. Both logistic regression and an artificial neural network model (Relevant Input Processor Network—RIPNet) were used for predictive modeling. When compared against segmentation resulting from cluster analysis, the RIPNet models performed between 25 and 30% better in absolute terms. This translates to a 50% higher true positive rate over given levels of false positives. This work indicates that it is feasible to build an automated system of plaque detection using MRI and data mining.

Ex vivo identification of atherosclerotic plaque calcification by a 31P solid‐state magnetic resonance imaging technique

Calcified tissue is a common component of atherosclerotic plaques, and occurs most often in mature plaques. The process of calcification is a poorly understood risk factor that may contribute to a plaque's vulnerability to sudden rupture. In this study a solid-state imaging sequence, termed single-point imaging (SPI), was used to observe calcification directly in ex vivo atherosclerotic plaques. Standards were used to validate the ability of (31)P SPI to detect and differentiate calcification from crystalline cholesterol, phospholipids, and other plaque components. After suitable experimental parameters were found, human carotid specimens obtained by endarterectomy were imaged ex vivo by (31)P solid-state imaging and standard (1)H methods. In contrast to (1)H imaging methods, (31)P imaging detected only the calcification in the plaque.

Association of high-density lipoprotein levels and carotid atherosclerotic plaque characteristics by magnetic resonance imaging

A low level of high-density lipoprotein cholesterol (HDL-C) is a risk factor for atherosclerotic disease. Magnetic resonance imaging (MRI) can provide detailed information on carotid atherosclerotic plaque size and composition. The purpose of this study was to correlate HDL levels with carotid plaque burden and composition by MRI. Thirty-four patients with coronary artery disease (CAD) receiving simvastatin plus niacin or placebo for both drugs for three years were randomly selected to undergo MRI of carotid arteries. Atherosclerotic plaque wall volumes (WVs) and plaque components including lipid rich/necrotic core (LR/NC), calcium, fibrous tissue, and loose matrix were measured. Mean WV or atherosclerotic burden was significantly associated with total HDL-C levels (r = -0.39, P = 0.02), HDL(2) (r = -0.36, P = 0.03), HDL(3) (r = -0.34, P = 0.04), and LDL/HDL ratio (r = 0.42, P = 0.02). Plaque lipid composition or LR/NC was significantly associated with HDL(3) (r = -0.68, P = 0.02). Patients with low HDL levels (<or=35 mg/dL) had increased WV (97 +/- 23 vs. 81 +/- 19 mm(3), P = 0.05) compared with patients with HDL levels > 35 mg/dL. Among CAD patients, low HDL-C levels were significantly associated with increased carotid atherosclerotic plaque burden and lipid content by MRI.

In Vivo Characterization and Quantification of Atherosclerotic Carotid Plaque Components With Multidetector Computed Tomography and Histopathological Correlation

In a previous in vitro study we have demonstrated that atherosclerotic plaque components can be characterized with multidetector computed tomography (MDCT) based on differences in Hounsfield values (HV). Now we evaluated the use of MDCT in vivo to characterize and quantify atherosclerotic carotid plaque components compared with histology as reference standard. Fifteen symptomatic patients with carotid stenosis (>70%) underwent MDCT angiography before carotid endarterectomy (CEA). From each CEA specimen 3 histological sections and corresponding MDCT images were selected. The HV of the major plaque components were assessed. The measured HV were: 657+/-416HU, 88+/-18HU, and 25+/-19HU for calcifications, fibrous tissue, and lipid core, respectively. The cut-off value to differentiate lipid core from fibrous tissue and fibrous tissue from calcifications was based on these measurements and set at 60 HU and 130 HU, respectively. Regression plots showed good correlations (R2>0.73) between MDCT and histology except for lipid core areas, which had a good correlation (R2=0.77) only in mildly calcified (0% to 10%) plaques. MDCT is able to quantify total plaque area, calcifications, and fibrous tissue in atherosclerotic carotid plaques in good correlation with histology. Lipid core can only be adequately quantified in mildly calcified plaques.

Quantification of atherosclerotic plaque components using in vivo MRI and supervised classifiers

In this work we aimed to study the possibility of using supervised classifiers to quantify the main components of carotid atherosclerotic plaque in vivo on the basis of multisequence MRI data. MRI data consisting of five MR weightings were obtained from 25 symptomatic subjects. Histological micrographs of endarterectomy specimens from the 25 carotids were used as a standard of reference for training and evaluation. The set of subjects was divided in a training set (12 subjects) and an evaluation set (13 subjects). Four different classifiers and two human MRI readers determined the percentages of calcified tissue, fibrous tissue, lipid core, and intraplaque hemorrhage on the subject level for all subjects in the evaluation set. Quantification of the relatively small amounts of calcium could not be done with statistical significance by either the classifiers or the MRI readers. For the other tissues a simple Bayesian classifier (Bayes) performed better than the other classifiers and the MRI readers. All classifiers performed better than the MRI readers in quantifying the sum of hemorrhage and lipid proportions. The MRI readers overestimated the hemorrhage proportions and tended to underestimate the lipid proportions. In conclusion, this pilot study demonstrates the benefits of algorithmic classifiers for quantifying plaque components.

3-D reconstruction of tissue components for atherosclerotic human arteries using ex vivo high-resolution MRI

Automatic computer-based methods are well suited for the image analysis of the different components in atherosclerotic plaques. Although several groups work on such analysis some of the methods used are oversimplified and require improvements when used within a computational framework for predicting meaningful stress and strain distributions in the heterogeneous arterial wall under various loading conditions. Based on high-resolution magnetic resonance imaging of excised atherosclerotic human arteries and a series of two-dimensional (2-D) contours we present a segmentation tool that permits a three-dimensional (3-D) reconstruction of the most important tissue components of atherosclerotic arteries. The underlying principle of the proposed approach is a model-based snake algorithm for identifying 2-D contours, which uses information about the plaque composition and geometric data of the tissue layers. Validation of the computer-generated tissue boundaries is performed with 100 MR images, which are compared with the results of a manual segmentation performed by four experts. Based on the Hausdorff distance and the average distance for computer-to-expert differences and the interexpert differences for the outer boundary of the adventitia, the adventitia-media, media-intima, intima-lumen and calcification boundaries are less than 1 pixel (0.234 mm). The percentage statistic shows similar results to the modified Williams index in terms of accuracy. Except for the identification of lipid-rich regions the proposed algorithm is automatic. The nonuniform rational B-spline-based computer-generated 3-D models of the individual tissue components provide a basis for clinical and computational analysis.

Intravascular Optical Coherence Tomography: Comparison with Histopathology in Atherosclerotic Peripheral Artery Specimens

Intravascular optical coherence tomography (OCT) is a new imaging modality that provides microstructural information on atherosclerotic plaques and has an axial resolution of 10-20 microm. OCT of coronary arteries characterizes different atherosclerotic plaque components by their distinctive signal patterns. Peripheral human arteries were examined ex vivo by means of OCT, and attempts to distinguish among fibrous, lipid-rich, and calcified atherosclerotic plaques were made based on imaging criteria previously established for coronary arteries. One hundred fifty-one atherosclerotic arterial segments were obtained from 15 below-knee amputations. OCT imaging criteria for different plaque types (fibrous, lipid-rich, calcified) were established in a subset of 30 arterial segments. The remaining 121 OCT images were analyzed by two independent readers. Each segment was divided into four quadrants. Agreement between histopathology and OCT was quantified by the kappa test of concordance, as were interobserver, intraobserver, and inter-method variability. Four hundred sixty-nine of 484 quadrants (97%) were available for comparison. Sensitivity and specificity for OCT criteria (consensus readers 1 and 2) were 86% and 86% for fibrous plaques, 78% and 93% for lipid-rich plaques, and 84% and 95% for calcified plaques, respectively (overall agreement, 84%). The interobserver and intraobserver reliabilities of OCT assessment were high (kappa values of 0.84 and 0.87, respectively). The inter-method agreement was 0.74 for consensus OCT versus consensus histology. OCT of peripheral human arteries ex vivo characterized different atherosclerotic plaque types with a high degree of agreement with histopathologic findings. Findings were comparable to those reported for coronary arteries. OCT promises to improve understanding of the progression or regression of peripheral atherosclerosis in vivo.

Young’s modulus reconstruction of vulnerable atherosclerotic plaque components using deformable curves

Rupture, with subsequent thrombosis, of thin-cap fibroatheromas (TCFAs) is a major cause of myocardial infarction. A TCFA has two main components: these are a large, soft lipid pool and a thin, stiff fibrous cap covering it. Quantification of their morphology and stiffness is essential for monitoring atherosclerosis and quantifying the effect of plaque-stabilizing pharmaceutical treatment. To accomplish this, we have developed a model-based Young’s modulus reconstruction method. From a plaque strain elastogram, measured with an intravascular ultrasound catheter, it reconstructs a Young’s modulus image of the plaque. To this end, a minimization algorithm automatically varies the morphology and stiffness parameters of a TCFA computer model, until the corresponding computer-simulated strain elastogram resembles the measured strain elastogram. The morphology parameters of the model are the control-points of two deformable Bézier curves; one curve delineates the distal border of the lipid pool region, the other the distal border of the cap region. These component regions are assumed to be homogeneous and their stiffness is characterized by a Young’s modulus. Reconstructions from strain elastograms that were 1. simulated using a histology-derived computer TCFA, 2. measured from a physical phantom with a soft lipid pool, and 3. simulated with a computer TCFA, where the complexity of its plaque component borders was increased, demonstrated the superior reconstruction/delineation behavior of this method, compared with a previously developed circular reconstruction method that used only circles for border delineation. Consequently, this method may become a valuable tool for the quantification of both the morphology and stiffness of vulnerable atherosclerotic plaque components. (E-mail: r.baldewsing@erasmusmc.nl)

Bright and black blood imaging of the carotid bifurcation at 3.0 T

Purpose The aim of this study was to evaluate our preliminary experience at 3.0 T with imaging of the carotid bifurcation in healthy and atherosclerotic subjects. Application at 3.0 T is motivated by the signal-to-noise gain for improving spatial resolution and reducing signal averaging requirements. Materials and methods We utilized a dual phased array coil and applied 2D, 3D time of flight (TOF) and turbo spin echo (TSE) sequences with comparison of two lumen signal suppression methods for black blood (BB) TSE imaging including double inversion preparation (DIR) and spatial presaturation pulses. The signal-to-noise ratios (SNR) of healthy carotid vessel walls were compared in 2D and 3D BB TSE acquisitions. The bright and black blood multi-contrast exam was demonstrated for a complex carotid plaque. Results Contrast-to-noise (CNR) greater than 150 was achieved between the lumen and suppressed background for 3D TOF. For BB, both methods provided sufficient lumen signal suppression but slight residual flow artifacts remained at the bifurcation level. As expected 3D TSE images had higher SNR compared to 2D, but increased motion sensitivity is a significant issue for 3D at high field. For multi-contrast imaging of atherosclerotic plaque, fibrous, calcified and lipid components were resolved. The CNR ratio of fibrous (bright on PDW, T2W) and calcified (dark in T1W, T2W, PDW) plaque components was maximal in the T2W images. The 3D TOF angiogram indicating a 40% stenosis was complemented by 3D multi-planar reformat of BB images that displayed plaque extent. Detection of intimal thickening, the earliest change associated with atherosclerotic progression was observed in BB PDW images at 3.0 T. Conclusions High SNR and CNR images have been demonstrated for the healthy and diseased carotid. Improvements in RF coils along with pulse sequence optimization, and evaluation of endogenous and exogenous contrast mechanisms will further enhance carotid imaging at 3.0 T.

Recent trends in coronary intravascular ultrasound: Tracking atherosclerosis, pursuit of vulnerable plaques, and beyond

Within the past decade, intravascular ultrasound (IVUS) has proved to be a useful modality for guiding coronary interventions and assessment of cardiac allograft vasculopathy. During the last few years, IVUS has been used increasingly for the evaluation of the efficacy of various possible antiatherosclerotic therapies including high-dose statins, antihypertensives, and recombinant apolipoprotein A-I Milano. The recently introduced radiofrequency signal analysis is an advanced method for evaluation of IVUS signals to determine various histologic components of atherosclerotic plaques. Elastography is an allied technology for measuring the tissue strain that occurs in response to changes in intraluminal pressure. Whether radiofrequency signal analysis or elastography will become clinically useful tools for detecting vulnerable plaques remains an unanswered, burning question.

Mo-W10:6 The atherosclerotic plaque components lysophosphatidic acid and collagen synergistically activate platelets

Mo-W10:6 The atherosclerotic plaque components lysophosphatidic acid and collagen synergistically activate platelets

Association of Cholesterol Subfractions and Carotid Lipid Core Measured by MRI

To the Editor: A strong relationship exists between cholesterol and atherosclerosis,1 with low density lipoprotein (LDL) being a major risk factor.2 However, 50% of patients with acute coronary events have “normal” cholesterol, and 75% patients with premature coronary heart disease (CHD) have normal LDL.3 Thus, contribution of other lipoproteins has been explored. High density lipoprotein (HDL), comprised primarily of 2 subfractions, HDL2 (large buoyant) and HDL3 (small dense), has a protective role in CHD.4,5 LDL can be dense or buoyant, and dense LDL is highly atherogenic, associated with 4-fold increased CHD risk.6 Lipoproteina [Lp(a)], is a strong risk factor for CHD and stroke.7 MRI can noninvasively visualize arterial wall remodelling and atherosclerotic plaque components (lipid core, fibrous cap, and calcium).8,9 However, in vivo relationships between plaque components and cholesterol subfractions have not been demonstrated in humans. We examined in vivo relationship between cholesterol subfractions and atherosclerotic plaque, measured by MRI, in internal carotid arteries (ICA) of atherosclerotic patients. This was an exploratory cross-sectional study of consecutively enrolled initial 28 patients who were part of an ongoing randomized trial testing the effects of extended release Niacin versus placebo station top of baseline therapy on carotid plaque regression, analyzed at baseline. All patients signed written informed consent and had documented atherosclerosis in …

HMGA1 proteins in human atherosclerotic plaques

One of the major characteristics of atherosclerosis is the migration of smooth muscle cells (SMC) from the tunica media to the intima, caused by alterations in the environment, e.g. mechanical, chemical, or immunologic injuries of the arterial walls. A group of molecules that may act as a main regulator of SMC phenotype switching is formed by the so-called HMGA1 high-mobility group proteins. One target gene of the HMGA1 protein, playing a major role in the development of atherosclerotic lesions, is CD44. The expression of CD44 is regulated by IL-1 β , but binding of HMGA1 potentiates the transactivation of the CD44 promoter. In this study, the HMGA1 expression of human atherosclerotic plaque samples was examined. Compared to the non-active components, all major components of the well-developed atherosclerotic plaques showed strong positivity of the high-mobility group protein HMGA1 in their activated areas, e.g. neointimal SMCs, macrophages, newly built blood vessels. This report is the first to describe HMGA1 as one of the first mediators in the development of human atherosclerotic plaques.

Assessment of Human Atherosclerotic Carotid Plaque Components with Multisequence MR Imaging: Initial Experience

To prospectively determine, by using a stepwise logistic regression model, the optimal magnetic resonance (MR) weighting (ie, pulse sequence) combinations for plaque assessment and corresponding cutoff values of relative signal intensities (rSIs). Institutional review board approval and patient consent were obtained. Eleven patients (seven men, four women; mean age +/- standard deviation, 68 years +/- 4) with symptomatic carotid disease and stenosis of more than 70% were investigated at MR imaging before carotid endarterectomy. The MR images were matched with histologic features of the endarterectomy specimens (reference standard). The rSIs (compared with that of muscle tissue) from regions of interest were assessed qualitatively and semiquantitatively. For all major components (calcification, lipid core, intraplaque hemorrhage, and fibrous tissue), optimal cutoff points for the rSIs were determined for five MR weightings by means of receiver operating characteristic curves. The best predicting combinations of these five dichotomized MR weightings were selected by means of stepwise logistic regression analysis. The potential sensitivity and specificity of MR imaging for vulnerable plaque with hemorrhage and/or lipid core were determined. The same optimal MR weighting combinations for identifying the four plaque components were found with qualitative and semiquantitative analysis. Sensitivity and specificity for vulnerable plaque were 93% (95% confidence interval: 77%, 99%) and 96% (95% confidence interval: 86%, 100%), respectively, for the qualitative analysis and 76% (95% confidence interval: 56%, 90%) and 100% (95% confidence interval: 93%, 100%) for the semiquantitative analysis. This study demonstrates the potential of a systematic approach of atherosclerotic plaque assessment with multisequence MR imaging by using the information provided from five different MR weightings in a stepwise logistic regression model.

Plasma delipidation process induces rapid regression of atherosclerosis and mobilisation of adipose tissue

Cholesterol is a major component of atherosclerotic plaques. Cholesterol accumulation within the arterial intima and atherosclerotic plaques is determined by the difference of cellular cholesterol synthesis and/or influx from apo B-containing lipoproteins and cholesterol efflux. In humans, apo A-1 Milano infusion has led to rapid regression of atherosclerosis in coronary arteries. We hypothesised that a multifunctional plasma delipidation process (PDP) would lead to rapid regression of experimental atherosclerosis and probably impact on adipose tissue lipids. In hyperlipidemic animals, the plasma concentrations of cholesterol, triglyceride and phospholipid were, respectively, 6-, 157-, and 18-fold higher than control animals, which consequently resulted in atherosclerosis. PDP consisted of delipidation of plasma with a mixture of butanol-diisopropyl ether (DIPE). PDP removed considerably more lipid from the hyperlipidemic animals than in normolipidemic animals. PDP treatment of hyperlipidemic animals markedly reduced intensity of lipid staining materials in the arterial wall and led to dramatic reduction of lipid in the adipose tissue. Five PDP treatments increased apolipoprotein A1 concentrations in all animals. Biochemical and hematological parameters were unaffected during PDP treatment. These results show that five PDP treatments led to marked reduction in avian atherosclerosis and removal of lipid from adipose tissue. PDP is a highly effective method for rapid regression of atherosclerosis.

Plasma Matrix Metalloproteinase-8 Concentrations are Associated With the Presence and Severity of Coronary Artery Disease

Interstitial collagen, especially type I, is a major component of atherosclerotic plaques and the matrix metalloproteinases (MMP) 1, 8 and 13 can initiate collagen breakdown. MMP-8 degrades type I collagen preferentially and more potently than MMP-1 or MMP-13. Although MMP-8 was thought to be produced only by neutrophils, it was recently reported to also be produced by endothelial cells, smooth muscle cells and macrophages in plaques. Plasma MMP-8 concentrations were measured in 250 patients undergoing coronary angiography for coronary artery disease (CAD: >50% stenosis), which was found in 181 patients, of whom 69 had 1-vessel, 66 had 2-vessel, and 46 had 3-vessel disease. Compared with 69 patients without CAD, the 181 with CAD had higher MMP-8 concentrations (3.5 vs 3.0 ng/ml, p < 0.001). There was a stepwise increase in MMP-8 concentration depending on the number of stenotic vessels: 3.2 in 1-vessel, 3.6 in 2-vessel, and 4.3 ng/ml in 3-vessel disease (p < 0.001). Multivariate analysis showed that MMP-8 concentration was independently associated with CAD. The odds ratio for CAD was 1.22 (95%confidence interval = 1.07-1.39) for a 1 ng/ml increase in MMP-8 concentration. Plasma MMP-8 concentration is associated with the presence and severity of CAD.