Quantification of Calcification in Atherosclerotic Lesions
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动脉硬化血栓血管生物学 2005年第8期
From the Departments of Medicine and Biochemistry/Molecular Biology, Baylor College of Medicine, Houston, Tex.
Correspondence to Dr Joel D. Morrisett, The Methodist Hospital, A601, 6565 Fannin St, Houston, TX 77030. E-mail morriset@bcm.tmc.edu
Abstract
Calcification can be deposited throughout the vasculature in several forms of calcium phosphate, including calcium hydroxyapatite (CHA). Calcium accumulation in arteries by mineralization and calcium loss from bone by osteoporosis often coexist, and vascular calcification may share common mechanisms with bone remodeling. Deposition of calcification in valves and arteries diminishes the valvular or arterial wall elasticity, a major cause of aneurysm and stenosis. Obstruction of arteries by calcification and other components can lead to heart attack and stroke. Mineralization in the femoral arteries can cause intermittent claudication in the legs, causing decreased mobility. Accurate measurement of calcification is essential for identifying other factors associated with this process and ultimately for elucidating the mechanism(s) of calcification. A wide range of methods for visualizing and measuring calcification for diagnosis and treatment in vivo and for studying the calcification process ex vivo are available. This review provides a critical comparison of older established methods and newer evolving technologies for quantifying calcification.
Key Words: calcification ? atherosclerosis ? MRI, micro-computed tomography ? ultrasound
Introduction
Calcium and phosphorus are the most abundant minerals in the body and are involved in a wide range of biochemical pathways, but mostly in the formation of calcium hydroxyapatite (CHA; Ca10(PO4)6(OH)2). Although CHA is the major natural component of bones and teeth, it can also deposit along with other forms of calcium phosphate in the vasculature with adverse affects. This type of deposit, known as vascular calcification, can ultimately lead to blood vessel stenosis, ischemia, and death. Approximately 90% of patients with cardiovascular disease (CVD) have vascular calcification. Because CVDs are the leading cause of death in the United States, there is considerable interest in understanding the mechanism(s) of vascular calcification and the implications for CVD.
Healthy bone exists in a dynamic state of remodeling, requiring osteoblasts that build bone under alkaline conditions and osteoclasts that degrade it under acidic conditions.1 Cytokines, such as bone morphogenetic proteins (BMPs), interleukin-6, insulin-like growth factor-1, as well as various hormones, regulate bone remodeling.2 As a consequence of remodeling, bone calcium and phosphate turnover occurs. In this turnover process, resorbed minerals are used to regenerate bone. When the calcium lost in degradation exceeds the calcium deposited in remodeling, there is a net loss of bone mass. With increasing age, the rate of bone degradation exceeds the rate of formation, resulting in osteoporosis, particularly in postmenopausal women, with decreased estrogens, which inhibit cytokines.3 Some of the calcium and phosphate mobilized by remodeling may become deposited in the arterial wall, leading to atherosclerosis in 1 arterial bed.
CHA is the most stable form of insoluble calcium phosphate.4 Under biological conditions, formation of CHA proceeds through noncrystalline amorphous calcium phosphate (ACP), which is stable under alkaline conditions (pH 8). Formation of ACP and CHA can be inhibited by many ions and other factors at their normal tissue concentrations. Osteoblast activity regulates formation of CHA, and alkaline phosphatase, which operates under alkaline conditions, is a marker for formation of ACP and its conversion to CHA.4
Although the mechanism of nucleation of ACP and its conversion into CHA in bone mineralization is not entirely clear, some factors of the process are known. ACP is isothermally metastable compared with the more ordered CHA. Initial mineral deposits are associated with membrane vesicles and specific bone-associated proteins such as osteonectin, osteocalcin, and matrix -carboxy glutamate (Gla) protein.2,5 On nucleation, growth of CHA crystals requires increased concentrations of Ca2+ and PO43– ions.6 The transformation of ACP to CHA in vitro occurs at slightly alkaline pH (7.4 to 7.8) and is temperature and time dependent, suggesting that the process is autocatalytic.7
Presumably, decomposition of CHA in bone to its constituent ions follows the reverse process. Osteoclast activity, inhibited by increased Ca2+ concentrations, involves resorption and phagocytosis of calcium phosphate in an environment made acidic by carbonic anhydrase.8,9 Osteoclasts form tunnel capillaries into the bone.1 Interfacing with the bone surface is a ruffled border, one of the domains into which the osteoclast plasma membrane is divided.10 Formation of a sealed compartment between the ruffled border and bone surface results in an acidic compartment in which CHA crystals disintegrate.4 Subsequently, the crystals are fragmented, transported through the osteoclast by a transcytotic vesicle, and exported to the vasculature.10
Vascular calcification is a well-ordered, regulated process similar to mineralization of bone tissue.11 Modulation of this process includes apoptosis of vascular smooth muscle cells (VSMCs), cell–cell interactions (macrophages and VSMCs), lipids, and plasma inorganic phosphate (Pi) levels.2 Four types of vascular calcification have been identified: atherosclerotic (fibrotic), cardiac valve, medial artery, and vascular calciphylaxis.2 In the atherosclerotic type, calcification initially occurs in the necrotic core of the plaque, and these types of lesions typically occur in or near bifurcations of arteries.2 Mechanical stressors and inflammation exacerbate cardiac valve calcification. Medial artery calcification often occurs in the femorals and is characteristic of diabetes and end-stage renal disease.2 Vascular calciphylaxis, or soft tissue calcification, is associated with a serum calcium–phosphate solubility product (Ksp) >60 mg2/dL2.2 The extent of calcification is strongly associated with stroke, amputation, and cardiovascular mortality.2
Calcification in atherosclerotic lesions involves factors important for bone mineralization, including matrix vesicles, BMP-2, osteopontin, osteocalcin, and collagen I.2 However, a major difference between vascular calcification and bone mineralization is the presence of oxidized lipids in the former but not the latter.12 The accumulation of oxidized lipids in the subendothelial space of arteries promotes arterial calcification, whereas these lipids in skeletal bone inhibit bone formation, suggesting another link between osteoporosis and vascular calcification.13
Osteoprotegerin, which protects against osteoporosis, forms a perimeter around calcified lesions.14 This protein is also in equilibrium with receptor activator of nuclear factor kappa and receptor activator of nuclear factor kappa ligand, which regulate the transition of preosteoclasts to fully differentiated osteoclasts.15 Several current investigations are focused on elucidating the mechanism(s) of vascular calcification. An important aspect of the task is quantification of arterial calcification.
The major objective of this review is to critically evaluate the diversity of methods available for quantifying calcification by in vivo and ex vivo methods (Table). Whereas in vivo methods are used primarily for clinical assessment and treatment of CVD, ex vivo methods are critical to mechanistic studies. Among ex vivo analytical methods, cadaveric carotid arteries (CCAs) and carotid endarterectomy (CEA) tissues have been studied for measuring calcium content. The wide variety of approaches enables comprehensive study of calcification quantification.
Comparison of Methodologies for Calcification Quantitation
In Vivo Analysis
Detecting lesions in vivo has clinical importance for diagnosis and treatment as well as in research. These in vivo methods of quantifying calcification will be discussed with regard to their methodology, practice, capabilities, and limitations.
Ultrasonography
Ultrasonography (US) involves the transmission of high-frequency sound waves (2 to 10 MHz) through an anatomic site of interest followed by conversion of echoes into electrical impulses, producing 2D images. Different modes and types of US are used in echocardiography: brightness or B-mode, motion or M-mode, spectral Doppler, and color flow mapping.
B-mode US produces a gray-scale image with good anatomic detail of the ventricular septum, ventricular free walls, heart valves, papillary muscle, and chordae tendineae. Using B-mode US to evaluate carotid plaques, weak reflections (echolucent) have been associated with a higher risk of neurological events than plaques giving strong reflections (echorich).16 Echolucent plaques have higher content of lipid and hemorrhage than echorich plaques, which usually contain more calcification and fibrous tissue.16 Arterial ulceration is sometimes assigned incorrectly to pits in fibrotic plaque, 2D calcification with shadowing, atheromatous debris, or ulcerated plaque hemorrhage.17 B-mode US has been used extensively to determine carotid intimal-medial far-wall thickness18–20 and calcification. Plaque calcification can be identified by a bright, hyperechogenic area resulting in cone-shaped echo shadowing. Such qualitative imaging was used in a study of calcification in atherosclerotic plaques in association with polymorphisms of the human matrix Gla protein (MGP) gene, which codes for a protein that inhibits calcification by strongly binding calcium ions to its Gla residues.21 Significantly, calcification has been found to be prevalent in femoral atherosclerotic plaques of patients carrying a particular MGP allele, but this association does not apply for carotid plaques. This study demonstrates the use of B-mode US in locating calcification; however, this method does not allow for reliable quantitation of calcification because the image resolution does not allow for accurate delineation of plaque components (Table).
Intravascular ultrasound (IVUS) is an invasive method that details the relationship between plaque and vessel wall in real time throughout the coronary artery tree. The invasive nature of IVUS allows exact definition of not only the quantity but also the distribution of calcification within the vessel wall and the ability to classify different plaque substructures, helping to clarify ambiguous angiograms and delineate the exact nature of luminal encroachment. The central positioning of a high-frequency transducer within the target vessel facilitates high resolution of the arterial lumen-wall border, permitting a more precise definition of small ulcerations than is available by other diagnostic methods.22 However, this method cannot be used routinely until the pathologic significance of plaque ulceration is clearly defined, thereby avoiding possible disturbance of an unstable plaque.22 This method also enables identification of lesion subsets that may have an important natural history in development of atherosclerosis.23,24 Four types of atherosclerotic plaque components can be distinguished using IVUS: (1) lipid-rich (hypoechoic), (2) fibromuscular (soft echoes), (3) fibrous (bright echoic), and (4) calcific (bright echoes with shadowing behind the lesion).25 IVUS has been used to differentiate these components in several studies in vivo.26 The precision and accuracy of calcification quantitation by IVUS is excellent.27 In a study of calcium in culprit lesions after the placement of a stent, IVUS was used to assess the arc of calcium, which appears as a bright echogenic signal accompanied by an acoustic shadow in the arterial wall.28 Calcium was quantified by 2 methods: (1) as the widest arc of calcium found in the stented segment, and (2) as the average arc of calcium in the proximal, middle, and distal sections of the stented segments. The results showed that calcium is less abundant in plaques associated with culprit stenoses and more abundant in plaques associated with stable angina.
Similarly, the calcium arc was used to quantify calcium in a study of coronary artery remodeling.29 Overall, coronary arteries were observed to enlarge in most patients, with the exception of smokers. Also, no specific morphological features were found to be predictive of arterial remodeling. In another study focused on determining the relationship between smoking and calcification, the arc of calcium as determined by IVUS was applied to a regression equation that took into account age, gender, and smoking.30 The results indicated that among patients with coronary artery disease, previous or current smokers have plaque areas with similar dimensions but less calcification than nonsmokers.
The method of measuring calcium content from IVUS images involves a process of gathering cross-sectional images at a reference site and at a plaque site to measure the arc of calcium.31 These measurements take into account the amount of calcium versus the lumen surface size by converting the calcium arc to a percentage of the lumen surface, producing a more accurate measure of calcification.
US tissue characterization coupled with integrated backscatter (IB) analysis is effective in distinguishing lipidic, fibrotic, and calcific components in human atherosclerotic plaques.32 This technique is capable of producing 2D images and IB images, the latter of which details biochemical and structural components of atherosclerotic lesions.33 In conjunction with IVUS, IB data have been used to make color-coded maps of coronary arterial plaques according to 5 tissue components: lipid core with fibrous cap, intimal hyperplasia, fibrous tissue, calcification, and thrombus.33,34
The most commonly used method to identify or quantify calcification by US is IVUS (Table). However, combinations of US methods are also incorporated but are used primarily for visualization purposes, not quantifying calcification.35–38 To determine the role of calcium–phosphate metabolism in cardiac valve calcification of hemodialysis patients, B-mode and Doppler US were used to image the areas of calcification and to determine the severity score of valvular calcifications on the basis of thickness.39 These scores were found to correlate with the calcium–phosphate product ([Ca]x[PO4]) calculated from the serum concentrations of the individual atoms.
Electron Beam CT
CT is based on x-ray technology that computes axial images of the body. In standard CT scanning of the heart, multiple cross-sectional images are acquired from different angles. A 3D view of the heart is then created by compiling the axial images. Although CT renders high-resolution images of still objects, it is not fast enough to acquire such images of a beating heart; however, it does allow noninvasive detection and reproducible quantification of calcification in vivo (Table).
Electron beam CT (EBCT) has been used to determine the presence and amount of calcium accumulated in the coronary arteries. It is much faster than standard CT scanning, produces images in a fraction of a second, and acquires high-resolutions images of an artery even while the heart is beating. EBCT uses an electron beam in stationary tungsten targets, permitting very rapid scanning times.40 Prospective electrocardiographic triggering is required for acquisition of images by EBCT to reduce cardiac motion artifacts. As a result, arterial fat and calcium accumulation can be visualized clearly by EBCT. In 100 milliseconds, serial transaxial images are obtained every 3 to 6 mm for purposes of detecting coronary artery calcium. Thinner sections have been found to provide more accurate results.41,42 Current EBCT software permits quantification of calcium area and density. The images produced provide the basis for a patient’s "calcium score," representing the total amount of calcium present in the artery and calculated using the following equation: calcium score=[sum of (suprathreshold areaxN)]xT/3, in which N is a density index with a value of 1 to 4 based on a truncated peak CT number (a measure of density with a range of 130 to 499) and T is the slice thickness.43 A calcium score of 0 indicates virtually no risk for a cardiac event; scores between 1 and 100 correlate with low risk for a cardiac event over the next 5 years; scores from 100 to 400 infer moderate risk for cardiac events; and scores >400 indicate high risk for a heart attack.40 Although this cardiac risk classification is not scientifically validated, it is useful as a clinical method of diagnosis and prognosis of heart disease. Another system of quantifying calcification as analyzed by CT is the Agatston score, which can be calculated using the number, area, and peak Hounsfield numbers of the detected calcified lesions and is based on 3-mm slices acquired without overlap.44 Volume and mass calculations also provide reproducible results. Additionally, percentiles of risk stratification have been suggested.45
EBCT provides an accurate, reliable alternative to the more commonly used stress tests but is more expensive than many comparable tests. Results of EBCT have been compared with those of 2D echocardiography,46 Doppler US,47 IVUS,48 and angiography48–50 and have been found to be highly accurate in localizing and quantifying calcifications in the heart. EBCT is also very effective for detecting stable calcification in the arterial wall, a feature highly correlated to age.51
An alternative method to EBCT is multidetector row spiral CT (MDCT) with electrocardiography gating.52–56 MDCT allows image acquisition of thinner slices but requires higher radiation exposure. Quantification of calcification by MDCT is more accurate than by EBCT or IVUS. Noncalcified plaque can also be visualized by MDCT but requires the aid of injected contrast-enhancing dyes, and individual plaque components cannot be distinguished and quantified. Studies have been conducted to understand the differences in signal between stable and unstable angina57,58 and within plaques to differentiate composition,59 but CT is not yet a reliable source for quantifying calcification relative to other plaque components.
Magnetic Resonance Imaging
MRI is a powerful imaging technique that can produce images of anatomic structures and organs inside the body and often provides more spatial and contrast resolution than other imaging techniques such as CT, which requires x-ray images and the injection of a contrast dye. MRI is most effective at providing images of tissues or organs that contain water or lipid but is not as useful for imaging structures that contain rather low levels of these molecules, especially in the solid state. The sensitivity of in vivo images is enhanced by the use of phased array surface coils that can be placed near the anatomic site of interest.
MRI is capable of distinguishing various components of atherosclerotic plaques, such as fibrous tissue, lipids, calcification, and thrombus60–62 (Figure 1A). This capability enables determining lesion type and monitoring the progression and regression of atherosclerotic plaques (Table). Calcification typically appears dark in standard T1-, T2-, and proton density–weighted (PDW) images (Figure 1A) and, hence, can be difficult to distinguish from the lumen of a black blood image (Figure 1C).
Figure 1. A, Three axial imaging sequences performed to generate different contrast weightings at each slice location: PDW, T1W, and T2W MR images of cadaveric arteries. Feature space analysis allows for quantitation of plaque components. TR indicates repetition time; TE, echo time; ETL, echo train length; FSE, fast spin-echo; FOV, field of view. B, Plotting intensities of pixels at the same x-y address in the T1W, T2W, and PDW images generates a feature space plot containing multiple clusters; each cluster corresponds to a component of the tissue: calcification, lumen, intima, lipid, necrotic core, and media. Each cluster was partially identified by imaging separate tissue components isolated by microdissection. Integration of the cluster volumes gives a quantitative measure of the corresponding component. (C. Karmonik and J. Morrisett, unpublished results, 2005). C, MRI axial slices (3 mm) of the left carotid artery of a 67-year-old woman presenting with >60% stenosis. The slices begin 9 mm below the bifurcation (C3) and extend 6 mm above it into the internal branch (I2). The compositional heterogeneity of the vessel wall and partially occluded lumen is evident. Slice C3 has a patent lumen, but disease of the wall is apparent from the darker area (calcification) at 7:00 (white arrow). This feature becomes more prominent in slice C2, extending from 6:00 to 12:00 (white arrows); concentric bright bands (fibrous cap and lipid core) are seen from 2:00 to 6:00 (red arrows). Slice C1 contains an indentation indicating the beginning of the flow divider (white arrow); the beginning of the orifice of the partially occluded internal branch is evident. Slice B cuts directly through the bifurcation, clearly showing a rather patent external carotid (white arrow) but a highly stenosed internal carotid (red arrow); the occluding plaque exhibits intermediate brightness typical of lipid rich lesions. Slice I1 shows complete separation of the 2 branches; the external carotid (white arrow) has virtually no disease, whereas the internal carotid shows significant wall thickening at 4:00 to 7:00 (red arrows). The spatial and contrast resolution of these representative images are essential for the separation and quantification of plaque components.
In a study of carotid artery calcification and white matter ischemia, calcification was graded using 2 methods: (1) extent of calcification based on degrees of the vessel circumference occupied by calcification, and (2) thickness of calcification.63 In a study of the effects of lipid-lowering drugs on atherosclerotic plaques, the investigators used MRI to determine the calcium cluster area and its percentage of the total plaque area.64 The other plaque components were quantified in the same way so that comparisons could be made. Patients treated with intensive lipid-altering therapy had significantly lower percentages of lipid and higher percentages of calcium than those who were untreated. However, changes in percentages of plaque components can be misleading because if the proportion of 1 component decreases (eg, lipid), then the proportion of the other components (eg, calcification) must necessarily increase if their masses do not change.
We are currently using MRI to quantify calcified atherosclerotic lesions in the superficial femoral artery. The resulting images are used to guide endovascular intervention such as stenting, bypass, or remote endarterectomy (J. Morrisett and A. Lumsden, unpublished results, 2005).
Ex Vivo Analysis
Whereas in vivo analysis of atherosclerotic plaques is useful for diagnosis and treatment, ex vivo analysis is instructive for understanding mechanisms of plaque formation and establishing the identity of features of plaque images obtained in vivo. Ex vivo studies have been performed on CCA and CEA tissues. Carotid arteries from human cadavers provide anatomic information about the medial and adventitial layers, which are not usually present in CEA tissues. This advantage is diminished partly by the alteration in CCA tissue structure because of formalin fixation, in contrast to CEA tissue, which is obtained fresh or stored in a cryoprotecting buffer at –20°C without loss of structure
Micro-CT
Micro-CT (μCT) is similar to CT in that x-ray images are acquired at multiple angles around the object followed by computation of its tomogram. Currently available μCT instrumentation is typically a compact, desktop x-ray system for nondestructive reconstruction of 3D tissue microstructure with high spatial resolution. With μCT, it is possible to: (1) obtain transmission shadow images of tissue; (2) reconstruct any cross-section of the complete 3D object microstructure; (3) calculate distance, surface area, and volume; (4) analyze density and porosity of an object; and (5) achieve 3D rendering and realistic visualization through animation of the reconstructed images (Table).
With μCT, 3D radioscopic image data are acquired rapidly and noninvasively to capture thin cross-sections. Because of the low-dose radiation used, mice and rats can be imaged serially by this method. The resulting data, which have spatial resolution of 20 μm, are used in reconstruction calculations to generate realistic 3D images and to calculate morphological parameters. A major application of μCT has been to quantify the 3D microstructure of bone and to provide quantitative information about its functionality, porosity, and mineral density, making the technique useful for early detection of various bone pathologies, including osteoporosis.65–68
μCT can also be used to study calcification of the arterial wall68,69 and enables nondestructive visualization and localization of CHA in very thin tissue slices, although it does not allow for delineation of other plaque components (Figure 2). Two morphological distributions are observed: (1) calcification nodules localized mainly in the necrotic core or luminal surface, and (2) calcification plates localized more to the medial layer, often extending around a substantial fraction of its circumference. Calibrating the μCT system with known amounts of CHA is done by scanning a 96-well microtiter plate having wells filled with different amounts of hydrated CHA. The sum of electron dense areas in successive slices of CHA in each well enables construction of a standard calibration curve that can be used for calculation of unknown samples.
Figure 2. μCT images of a CCA. Axial slice images (0.3 mm) reveal a large calcification plate (a and b) and small nodules (c) in the internal carotid but no calcification in the common carotid (d).
Magnetic Resonance Imaging
Ex vivo samples can be imaged by MRI using coils of different geometries. A solenoid coil (30-mm diameter) is convenient for imaging single samples at very high resolution. A phased array coil (6-cm width) is convenient for multiple samples at high resolution. MRI analysis of CEA specimens can be performed using the same sample holder but smaller sample tubes than used for CCA imaging.
Representative magnetic resonance (MR) images of CEA tissues are shown in Figure 1. Plaque components, including collagenous cap, necrotic core, hemorrhage, and calcification can be distinguished in MR images of plaques.70–72 These components can also be mapped according to their contrast weightings, which allow for integration of the components and quantification. Multicontrast-weighted MR images of carotid plaques have also been used to develop classification maps useful in distinguishing plaque components more effectively69 (Table).
In a study of left and right CCA, MRI and EBCT were used to determine arterial wall volume and calcification score.73 The results indicated that total wall volume and plaque calcification burden were similar for the left and right arteries, suggesting that atherosclerosis is a bilaterally symmetrical disease.
In PDW images of CEA tissues, regions of calcification appear dark (Figure 3b). When these tissues are embedded in paraffin, the calcified regions appear white (Figure 3a). Integration of the calcified regions visualized by each method provided 2 sets of integrated areas (MRI and embedded tissue imaging) that were highly correlated (R2=0.99; Figure 3c).
Figure 3. a, A CEA specimen was microdissected into segments 1.0 cm in length. Selected segments were fixed and embedded in paraffin by conventional methods. From each block, excess paraffin was lifted with a microtome until a full artery cross-section was exposed. The fixed tissue cross-sections were digitally photographed and contrast-manually enhanced with the NIH image analysis program ImageJ 1.30v. b, Each digital image was matched using eFilm Workstation 1.8.3 with the MRI image of the closest morphology. For each image, the total artery area, total lumen area, and total calcified area were circumscribed independently using freehand area selection in ImageJ and measured by automated integration. Percent calcification was then calculated for each image. c, Comparison of percent calcification in CEA specimens detected by MRI (b) and embedded tissue imaging (ETI; a; S. Marvel and J. Morrisett, unpublished results, 2005).
Three-D images have been obtained of human CCA using multicontrast-weighted fast spin-echo imaging.74 A cluster analysis technique called spatially enhanced cluster analysis objectively classified and quantified multicontrast MR images. The cluster technique divides data into groups with strong associations by iteratively minimizing a characteristic of the cluster. Using this technique, plaque components such as calcification can be differentiated by color and then quantified.
31P MR Spectroscopy
MR spectroscopy (MRS) is useful for quantifying nuclei in specific magnetic environments and has been used extensively to study biomolecules in isotropic solutions. Magic angle spinning (MAS) extends the power of MRS to include determining chemical and structural properties of anisotropic liquid crystalline and solid samples. The mineral content of bone can be quantified using 31P MRS because the phosphate of CHA is distinguishable from Pi, from phosphorylated metabolites dissolved in the cytosol, and from the polar head groups of phospholipids in membranes.75,76 Accordingly, 31P MAS MRS can be used for rapid quantification of CHA in atherosclerotic plaques.77,78
In a study of lipid phases and CHA deposits in human atherosclerotic lesions, a plaque with low lipid content (weak 13C MAS MRS signals) and extensive calcification (strong 31P MAS MRS signals) has been used to determine CHA content.77 After delipidation of the plaque, the 31P MAS MRS signal intensity showed no change, indicating that the 31P signal resulted from nonlipid phosphorus. Based on the intensity of the 31P MAS MRS peak, the phosphorus content could be calculated, followed by stoichiometric conversion to CHA content.
The integrated 31P signal intensity in plaques can be calibrated using synthetic CHA and chicken bone powder as reference compounds.78 Comparisons of 31P peak intensities showed that the chicken bone powder provided the best calibration, presumably because the synthetic CHA has a more ordered crystalline structure than the biological samples. This technique is somewhat destructive because delipidation leaves the tissue in a non-native state. 31P MAS MRS is generally restricted to ex vivo samples <1 cm3 (Table).
Tissue Digital Photography
Digital photographs of CEA specimens are useful for documenting tissue features lost during processing (eg, thrombus and calcification) for microscopy and can capture subtle textural and morphological features not detected by other techniques (Figure 4). Although differences in photographic color are useful for qualitatively distinguishing between calcified and lipid-rich regions, the contrast is not sufficient to accurately quantify the 2 types of plaque components. However, estimating calcium in these tissues becomes feasible when they are embedded in paraffin and digitally photographed (Figure 3). The total artery area, total lumen area, and total calcified area can be integrated and these areas used to calculate the percent calcification for each image. Although paraffin-thin sections frequently lose some of the calcified component during cutting, the remaining block has a smooth surface that facilitates quantitation (Table).
Figure 4. Digital photograph of a fresh CEA specimen (a) illustrating the presence of extensive calcification and thrombus in segment I3 (b). Thrombus appears as a dark red area, and calcification appears as a white–pink area. The shiny white areas are optical artifacts.
Histology
Conventional histology is used widely to analyze the structure of animal and plant tissues. Fixed and stained CEA specimens viewed by light microscopy are useful for identifying plaque components.79 For calcified tissues in paraffin blocks, cutting intact cross-sections can be difficult. Exemplary images from histological analysis are shown in Figure 5. Cutting calcification in frozen sections is even more challenging, usually requiring at least treatment with nitric acid or EDTA. However, these treatments cause calcium depletion and can reduce immunoreactivity of calcium-binding proteins. The percentages of calcification and other plaque components can be determined by area integration.80,81
Figure 5. From the internal branch of the left carotid artery, fibro-fatty lesions have been segmented and fixed in paraffin blocks. Then 10-μm histology sections prepared from original blocks were stained with: a, Oil Red O, to stain lipid purple, and nuclei brown; b, Van Kossa, to stain calcium salts black, nuclei red, and cytoplasm light pink; and c, trichrome, to stain muscle fibers red, collagen blue, and nuclei blue to black. These sections illustrate distribution of small calcification nodules through the midintimal area (heavy arrow) and loss of calcification from a deep-intimal area near 2:00 (light arrow).
Embedding tissue in plastic helps prevent loss of calcification during sectioning while retaining stain and antibody reactivity. Representative plastic embedded thin sections are shown in Figure 6. Paraffin blocks and frozen sections are attended by significant loss of calcification; however, the time-consuming process of tissue embedding in plastic allows retention of calcification and its quantification (Table).
Figure 6. X-ray image and corresponding thin-section light micrographs from right CCA fixed in 10% formalin. After fixation, the CEA samples are dehydrated in a graded series of ethanol washes and embedded in glycomethylmethacrylate. After polymerization, thin sections (10 to 30 um) are prepared using the Exakt System modified sawing microtome technique.84 Sections are stained with a Modified Goldner Tri-Chrome reagent for plastic sections for which calcification appears blue–green.
Chemical Analysis
Atomic absorption spectroscopy (AAS) is a sensitive analytical technique used to determine the concentration of metals in liquid samples. In their elemental form, metals absorb ultraviolet light when excited by heat, and each metal has a characteristic wavelength that will be absorbed. The amount of light absorbed is proportional to the concentration of the element in the solution. Measurements are made separately for each element of interest. The method is very sensitive and can measure trace elements down to the parts per million level. Because calcium is one of the metals detectable by AAS, this method is useful in determining calcium concentration in CEA samples. Stoichiometry can be used to calculate the quantity of CHA in the samples (Table).
Another method for chemically analyzing CHA in CEA samples uses the phosphomolybdate reagent, which quantifies the amount of elemental phosphorus in a sample.82 The sample is digested in sulfuric acid and oxidized with hydrogen peroxide to liberate elemental phosphorus. Ammonium molybdate and 1-amino-2-naphthol-4-sulfonic acid are added to produce a solution that absorbs at 830 nm. The concentration of phosphorus is determined from a calibration curve of various concentrations of a standard phosphate salt. The stoichiometry of CHA, Ca10(PO4)6(OH)2, can be used to convert the measured phosphorus concentration to CHA mass. A useful alternative method uses o-cresolphthalein for calcium determination.83 Fura and Indo ratiometric calcium indicators are excited with UV light, allowing for quantification of calcium content as well.84
Concluding Comments
Selecting an appropriate method of calcification quantification depends on the nature of the specimen and the desired results (Table). Although the in vivo methods of US, EBCT, and MRI are all capable of differentiating plaque components, IVUS and MRI have the highest spatial resolution. In ex vivo analyses, μCT and MRI are the principal methods for visualizing calcified thin slices and creating 3D reconstruction of the specimen. Digital photography is useful for capturing a visual record of intact tissues and tissue fragments before processing by embedding or homogenization. Histological sections provide morphological information that is currently the gold reference standard for validating many imaging techniques. 31P MAS MRS and the microphosphorus chemical assay for calcification quantification rely on chemical stoichiometry to convert the concentration of phosphorus atoms to CHA. The chemical assay is more amenable to high throughput but requires sample destruction. AAS is probably the most sensitive method for calcium quantification; it has the same capabilities and limitations as those of the microphosphorus assay. These qualitative and quantitative methods will likely see increasing use in future investigations of vascular calcification.
Acknowledgments
This work was supported in part by the National Institutes of Health grants HL63090 and HL07812. The authors thank Catherine Ambrose, PhD, and Tiffany Sheffield of the Bone Histomorphometry and Biomaterials Laboratory in the Department of Orthopaedic Surgery at the University of Texas-Houston Medical School for the plastic embedded histochemical preparations.
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Correspondence to Dr Joel D. Morrisett, The Methodist Hospital, A601, 6565 Fannin St, Houston, TX 77030. E-mail morriset@bcm.tmc.edu
Abstract
Calcification can be deposited throughout the vasculature in several forms of calcium phosphate, including calcium hydroxyapatite (CHA). Calcium accumulation in arteries by mineralization and calcium loss from bone by osteoporosis often coexist, and vascular calcification may share common mechanisms with bone remodeling. Deposition of calcification in valves and arteries diminishes the valvular or arterial wall elasticity, a major cause of aneurysm and stenosis. Obstruction of arteries by calcification and other components can lead to heart attack and stroke. Mineralization in the femoral arteries can cause intermittent claudication in the legs, causing decreased mobility. Accurate measurement of calcification is essential for identifying other factors associated with this process and ultimately for elucidating the mechanism(s) of calcification. A wide range of methods for visualizing and measuring calcification for diagnosis and treatment in vivo and for studying the calcification process ex vivo are available. This review provides a critical comparison of older established methods and newer evolving technologies for quantifying calcification.
Key Words: calcification ? atherosclerosis ? MRI, micro-computed tomography ? ultrasound
Introduction
Calcium and phosphorus are the most abundant minerals in the body and are involved in a wide range of biochemical pathways, but mostly in the formation of calcium hydroxyapatite (CHA; Ca10(PO4)6(OH)2). Although CHA is the major natural component of bones and teeth, it can also deposit along with other forms of calcium phosphate in the vasculature with adverse affects. This type of deposit, known as vascular calcification, can ultimately lead to blood vessel stenosis, ischemia, and death. Approximately 90% of patients with cardiovascular disease (CVD) have vascular calcification. Because CVDs are the leading cause of death in the United States, there is considerable interest in understanding the mechanism(s) of vascular calcification and the implications for CVD.
Healthy bone exists in a dynamic state of remodeling, requiring osteoblasts that build bone under alkaline conditions and osteoclasts that degrade it under acidic conditions.1 Cytokines, such as bone morphogenetic proteins (BMPs), interleukin-6, insulin-like growth factor-1, as well as various hormones, regulate bone remodeling.2 As a consequence of remodeling, bone calcium and phosphate turnover occurs. In this turnover process, resorbed minerals are used to regenerate bone. When the calcium lost in degradation exceeds the calcium deposited in remodeling, there is a net loss of bone mass. With increasing age, the rate of bone degradation exceeds the rate of formation, resulting in osteoporosis, particularly in postmenopausal women, with decreased estrogens, which inhibit cytokines.3 Some of the calcium and phosphate mobilized by remodeling may become deposited in the arterial wall, leading to atherosclerosis in 1 arterial bed.
CHA is the most stable form of insoluble calcium phosphate.4 Under biological conditions, formation of CHA proceeds through noncrystalline amorphous calcium phosphate (ACP), which is stable under alkaline conditions (pH 8). Formation of ACP and CHA can be inhibited by many ions and other factors at their normal tissue concentrations. Osteoblast activity regulates formation of CHA, and alkaline phosphatase, which operates under alkaline conditions, is a marker for formation of ACP and its conversion to CHA.4
Although the mechanism of nucleation of ACP and its conversion into CHA in bone mineralization is not entirely clear, some factors of the process are known. ACP is isothermally metastable compared with the more ordered CHA. Initial mineral deposits are associated with membrane vesicles and specific bone-associated proteins such as osteonectin, osteocalcin, and matrix -carboxy glutamate (Gla) protein.2,5 On nucleation, growth of CHA crystals requires increased concentrations of Ca2+ and PO43– ions.6 The transformation of ACP to CHA in vitro occurs at slightly alkaline pH (7.4 to 7.8) and is temperature and time dependent, suggesting that the process is autocatalytic.7
Presumably, decomposition of CHA in bone to its constituent ions follows the reverse process. Osteoclast activity, inhibited by increased Ca2+ concentrations, involves resorption and phagocytosis of calcium phosphate in an environment made acidic by carbonic anhydrase.8,9 Osteoclasts form tunnel capillaries into the bone.1 Interfacing with the bone surface is a ruffled border, one of the domains into which the osteoclast plasma membrane is divided.10 Formation of a sealed compartment between the ruffled border and bone surface results in an acidic compartment in which CHA crystals disintegrate.4 Subsequently, the crystals are fragmented, transported through the osteoclast by a transcytotic vesicle, and exported to the vasculature.10
Vascular calcification is a well-ordered, regulated process similar to mineralization of bone tissue.11 Modulation of this process includes apoptosis of vascular smooth muscle cells (VSMCs), cell–cell interactions (macrophages and VSMCs), lipids, and plasma inorganic phosphate (Pi) levels.2 Four types of vascular calcification have been identified: atherosclerotic (fibrotic), cardiac valve, medial artery, and vascular calciphylaxis.2 In the atherosclerotic type, calcification initially occurs in the necrotic core of the plaque, and these types of lesions typically occur in or near bifurcations of arteries.2 Mechanical stressors and inflammation exacerbate cardiac valve calcification. Medial artery calcification often occurs in the femorals and is characteristic of diabetes and end-stage renal disease.2 Vascular calciphylaxis, or soft tissue calcification, is associated with a serum calcium–phosphate solubility product (Ksp) >60 mg2/dL2.2 The extent of calcification is strongly associated with stroke, amputation, and cardiovascular mortality.2
Calcification in atherosclerotic lesions involves factors important for bone mineralization, including matrix vesicles, BMP-2, osteopontin, osteocalcin, and collagen I.2 However, a major difference between vascular calcification and bone mineralization is the presence of oxidized lipids in the former but not the latter.12 The accumulation of oxidized lipids in the subendothelial space of arteries promotes arterial calcification, whereas these lipids in skeletal bone inhibit bone formation, suggesting another link between osteoporosis and vascular calcification.13
Osteoprotegerin, which protects against osteoporosis, forms a perimeter around calcified lesions.14 This protein is also in equilibrium with receptor activator of nuclear factor kappa and receptor activator of nuclear factor kappa ligand, which regulate the transition of preosteoclasts to fully differentiated osteoclasts.15 Several current investigations are focused on elucidating the mechanism(s) of vascular calcification. An important aspect of the task is quantification of arterial calcification.
The major objective of this review is to critically evaluate the diversity of methods available for quantifying calcification by in vivo and ex vivo methods (Table). Whereas in vivo methods are used primarily for clinical assessment and treatment of CVD, ex vivo methods are critical to mechanistic studies. Among ex vivo analytical methods, cadaveric carotid arteries (CCAs) and carotid endarterectomy (CEA) tissues have been studied for measuring calcium content. The wide variety of approaches enables comprehensive study of calcification quantification.
Comparison of Methodologies for Calcification Quantitation
In Vivo Analysis
Detecting lesions in vivo has clinical importance for diagnosis and treatment as well as in research. These in vivo methods of quantifying calcification will be discussed with regard to their methodology, practice, capabilities, and limitations.
Ultrasonography
Ultrasonography (US) involves the transmission of high-frequency sound waves (2 to 10 MHz) through an anatomic site of interest followed by conversion of echoes into electrical impulses, producing 2D images. Different modes and types of US are used in echocardiography: brightness or B-mode, motion or M-mode, spectral Doppler, and color flow mapping.
B-mode US produces a gray-scale image with good anatomic detail of the ventricular septum, ventricular free walls, heart valves, papillary muscle, and chordae tendineae. Using B-mode US to evaluate carotid plaques, weak reflections (echolucent) have been associated with a higher risk of neurological events than plaques giving strong reflections (echorich).16 Echolucent plaques have higher content of lipid and hemorrhage than echorich plaques, which usually contain more calcification and fibrous tissue.16 Arterial ulceration is sometimes assigned incorrectly to pits in fibrotic plaque, 2D calcification with shadowing, atheromatous debris, or ulcerated plaque hemorrhage.17 B-mode US has been used extensively to determine carotid intimal-medial far-wall thickness18–20 and calcification. Plaque calcification can be identified by a bright, hyperechogenic area resulting in cone-shaped echo shadowing. Such qualitative imaging was used in a study of calcification in atherosclerotic plaques in association with polymorphisms of the human matrix Gla protein (MGP) gene, which codes for a protein that inhibits calcification by strongly binding calcium ions to its Gla residues.21 Significantly, calcification has been found to be prevalent in femoral atherosclerotic plaques of patients carrying a particular MGP allele, but this association does not apply for carotid plaques. This study demonstrates the use of B-mode US in locating calcification; however, this method does not allow for reliable quantitation of calcification because the image resolution does not allow for accurate delineation of plaque components (Table).
Intravascular ultrasound (IVUS) is an invasive method that details the relationship between plaque and vessel wall in real time throughout the coronary artery tree. The invasive nature of IVUS allows exact definition of not only the quantity but also the distribution of calcification within the vessel wall and the ability to classify different plaque substructures, helping to clarify ambiguous angiograms and delineate the exact nature of luminal encroachment. The central positioning of a high-frequency transducer within the target vessel facilitates high resolution of the arterial lumen-wall border, permitting a more precise definition of small ulcerations than is available by other diagnostic methods.22 However, this method cannot be used routinely until the pathologic significance of plaque ulceration is clearly defined, thereby avoiding possible disturbance of an unstable plaque.22 This method also enables identification of lesion subsets that may have an important natural history in development of atherosclerosis.23,24 Four types of atherosclerotic plaque components can be distinguished using IVUS: (1) lipid-rich (hypoechoic), (2) fibromuscular (soft echoes), (3) fibrous (bright echoic), and (4) calcific (bright echoes with shadowing behind the lesion).25 IVUS has been used to differentiate these components in several studies in vivo.26 The precision and accuracy of calcification quantitation by IVUS is excellent.27 In a study of calcium in culprit lesions after the placement of a stent, IVUS was used to assess the arc of calcium, which appears as a bright echogenic signal accompanied by an acoustic shadow in the arterial wall.28 Calcium was quantified by 2 methods: (1) as the widest arc of calcium found in the stented segment, and (2) as the average arc of calcium in the proximal, middle, and distal sections of the stented segments. The results showed that calcium is less abundant in plaques associated with culprit stenoses and more abundant in plaques associated with stable angina.
Similarly, the calcium arc was used to quantify calcium in a study of coronary artery remodeling.29 Overall, coronary arteries were observed to enlarge in most patients, with the exception of smokers. Also, no specific morphological features were found to be predictive of arterial remodeling. In another study focused on determining the relationship between smoking and calcification, the arc of calcium as determined by IVUS was applied to a regression equation that took into account age, gender, and smoking.30 The results indicated that among patients with coronary artery disease, previous or current smokers have plaque areas with similar dimensions but less calcification than nonsmokers.
The method of measuring calcium content from IVUS images involves a process of gathering cross-sectional images at a reference site and at a plaque site to measure the arc of calcium.31 These measurements take into account the amount of calcium versus the lumen surface size by converting the calcium arc to a percentage of the lumen surface, producing a more accurate measure of calcification.
US tissue characterization coupled with integrated backscatter (IB) analysis is effective in distinguishing lipidic, fibrotic, and calcific components in human atherosclerotic plaques.32 This technique is capable of producing 2D images and IB images, the latter of which details biochemical and structural components of atherosclerotic lesions.33 In conjunction with IVUS, IB data have been used to make color-coded maps of coronary arterial plaques according to 5 tissue components: lipid core with fibrous cap, intimal hyperplasia, fibrous tissue, calcification, and thrombus.33,34
The most commonly used method to identify or quantify calcification by US is IVUS (Table). However, combinations of US methods are also incorporated but are used primarily for visualization purposes, not quantifying calcification.35–38 To determine the role of calcium–phosphate metabolism in cardiac valve calcification of hemodialysis patients, B-mode and Doppler US were used to image the areas of calcification and to determine the severity score of valvular calcifications on the basis of thickness.39 These scores were found to correlate with the calcium–phosphate product ([Ca]x[PO4]) calculated from the serum concentrations of the individual atoms.
Electron Beam CT
CT is based on x-ray technology that computes axial images of the body. In standard CT scanning of the heart, multiple cross-sectional images are acquired from different angles. A 3D view of the heart is then created by compiling the axial images. Although CT renders high-resolution images of still objects, it is not fast enough to acquire such images of a beating heart; however, it does allow noninvasive detection and reproducible quantification of calcification in vivo (Table).
Electron beam CT (EBCT) has been used to determine the presence and amount of calcium accumulated in the coronary arteries. It is much faster than standard CT scanning, produces images in a fraction of a second, and acquires high-resolutions images of an artery even while the heart is beating. EBCT uses an electron beam in stationary tungsten targets, permitting very rapid scanning times.40 Prospective electrocardiographic triggering is required for acquisition of images by EBCT to reduce cardiac motion artifacts. As a result, arterial fat and calcium accumulation can be visualized clearly by EBCT. In 100 milliseconds, serial transaxial images are obtained every 3 to 6 mm for purposes of detecting coronary artery calcium. Thinner sections have been found to provide more accurate results.41,42 Current EBCT software permits quantification of calcium area and density. The images produced provide the basis for a patient’s "calcium score," representing the total amount of calcium present in the artery and calculated using the following equation: calcium score=[sum of (suprathreshold areaxN)]xT/3, in which N is a density index with a value of 1 to 4 based on a truncated peak CT number (a measure of density with a range of 130 to 499) and T is the slice thickness.43 A calcium score of 0 indicates virtually no risk for a cardiac event; scores between 1 and 100 correlate with low risk for a cardiac event over the next 5 years; scores from 100 to 400 infer moderate risk for cardiac events; and scores >400 indicate high risk for a heart attack.40 Although this cardiac risk classification is not scientifically validated, it is useful as a clinical method of diagnosis and prognosis of heart disease. Another system of quantifying calcification as analyzed by CT is the Agatston score, which can be calculated using the number, area, and peak Hounsfield numbers of the detected calcified lesions and is based on 3-mm slices acquired without overlap.44 Volume and mass calculations also provide reproducible results. Additionally, percentiles of risk stratification have been suggested.45
EBCT provides an accurate, reliable alternative to the more commonly used stress tests but is more expensive than many comparable tests. Results of EBCT have been compared with those of 2D echocardiography,46 Doppler US,47 IVUS,48 and angiography48–50 and have been found to be highly accurate in localizing and quantifying calcifications in the heart. EBCT is also very effective for detecting stable calcification in the arterial wall, a feature highly correlated to age.51
An alternative method to EBCT is multidetector row spiral CT (MDCT) with electrocardiography gating.52–56 MDCT allows image acquisition of thinner slices but requires higher radiation exposure. Quantification of calcification by MDCT is more accurate than by EBCT or IVUS. Noncalcified plaque can also be visualized by MDCT but requires the aid of injected contrast-enhancing dyes, and individual plaque components cannot be distinguished and quantified. Studies have been conducted to understand the differences in signal between stable and unstable angina57,58 and within plaques to differentiate composition,59 but CT is not yet a reliable source for quantifying calcification relative to other plaque components.
Magnetic Resonance Imaging
MRI is a powerful imaging technique that can produce images of anatomic structures and organs inside the body and often provides more spatial and contrast resolution than other imaging techniques such as CT, which requires x-ray images and the injection of a contrast dye. MRI is most effective at providing images of tissues or organs that contain water or lipid but is not as useful for imaging structures that contain rather low levels of these molecules, especially in the solid state. The sensitivity of in vivo images is enhanced by the use of phased array surface coils that can be placed near the anatomic site of interest.
MRI is capable of distinguishing various components of atherosclerotic plaques, such as fibrous tissue, lipids, calcification, and thrombus60–62 (Figure 1A). This capability enables determining lesion type and monitoring the progression and regression of atherosclerotic plaques (Table). Calcification typically appears dark in standard T1-, T2-, and proton density–weighted (PDW) images (Figure 1A) and, hence, can be difficult to distinguish from the lumen of a black blood image (Figure 1C).
Figure 1. A, Three axial imaging sequences performed to generate different contrast weightings at each slice location: PDW, T1W, and T2W MR images of cadaveric arteries. Feature space analysis allows for quantitation of plaque components. TR indicates repetition time; TE, echo time; ETL, echo train length; FSE, fast spin-echo; FOV, field of view. B, Plotting intensities of pixels at the same x-y address in the T1W, T2W, and PDW images generates a feature space plot containing multiple clusters; each cluster corresponds to a component of the tissue: calcification, lumen, intima, lipid, necrotic core, and media. Each cluster was partially identified by imaging separate tissue components isolated by microdissection. Integration of the cluster volumes gives a quantitative measure of the corresponding component. (C. Karmonik and J. Morrisett, unpublished results, 2005). C, MRI axial slices (3 mm) of the left carotid artery of a 67-year-old woman presenting with >60% stenosis. The slices begin 9 mm below the bifurcation (C3) and extend 6 mm above it into the internal branch (I2). The compositional heterogeneity of the vessel wall and partially occluded lumen is evident. Slice C3 has a patent lumen, but disease of the wall is apparent from the darker area (calcification) at 7:00 (white arrow). This feature becomes more prominent in slice C2, extending from 6:00 to 12:00 (white arrows); concentric bright bands (fibrous cap and lipid core) are seen from 2:00 to 6:00 (red arrows). Slice C1 contains an indentation indicating the beginning of the flow divider (white arrow); the beginning of the orifice of the partially occluded internal branch is evident. Slice B cuts directly through the bifurcation, clearly showing a rather patent external carotid (white arrow) but a highly stenosed internal carotid (red arrow); the occluding plaque exhibits intermediate brightness typical of lipid rich lesions. Slice I1 shows complete separation of the 2 branches; the external carotid (white arrow) has virtually no disease, whereas the internal carotid shows significant wall thickening at 4:00 to 7:00 (red arrows). The spatial and contrast resolution of these representative images are essential for the separation and quantification of plaque components.
In a study of carotid artery calcification and white matter ischemia, calcification was graded using 2 methods: (1) extent of calcification based on degrees of the vessel circumference occupied by calcification, and (2) thickness of calcification.63 In a study of the effects of lipid-lowering drugs on atherosclerotic plaques, the investigators used MRI to determine the calcium cluster area and its percentage of the total plaque area.64 The other plaque components were quantified in the same way so that comparisons could be made. Patients treated with intensive lipid-altering therapy had significantly lower percentages of lipid and higher percentages of calcium than those who were untreated. However, changes in percentages of plaque components can be misleading because if the proportion of 1 component decreases (eg, lipid), then the proportion of the other components (eg, calcification) must necessarily increase if their masses do not change.
We are currently using MRI to quantify calcified atherosclerotic lesions in the superficial femoral artery. The resulting images are used to guide endovascular intervention such as stenting, bypass, or remote endarterectomy (J. Morrisett and A. Lumsden, unpublished results, 2005).
Ex Vivo Analysis
Whereas in vivo analysis of atherosclerotic plaques is useful for diagnosis and treatment, ex vivo analysis is instructive for understanding mechanisms of plaque formation and establishing the identity of features of plaque images obtained in vivo. Ex vivo studies have been performed on CCA and CEA tissues. Carotid arteries from human cadavers provide anatomic information about the medial and adventitial layers, which are not usually present in CEA tissues. This advantage is diminished partly by the alteration in CCA tissue structure because of formalin fixation, in contrast to CEA tissue, which is obtained fresh or stored in a cryoprotecting buffer at –20°C without loss of structure
Micro-CT
Micro-CT (μCT) is similar to CT in that x-ray images are acquired at multiple angles around the object followed by computation of its tomogram. Currently available μCT instrumentation is typically a compact, desktop x-ray system for nondestructive reconstruction of 3D tissue microstructure with high spatial resolution. With μCT, it is possible to: (1) obtain transmission shadow images of tissue; (2) reconstruct any cross-section of the complete 3D object microstructure; (3) calculate distance, surface area, and volume; (4) analyze density and porosity of an object; and (5) achieve 3D rendering and realistic visualization through animation of the reconstructed images (Table).
With μCT, 3D radioscopic image data are acquired rapidly and noninvasively to capture thin cross-sections. Because of the low-dose radiation used, mice and rats can be imaged serially by this method. The resulting data, which have spatial resolution of 20 μm, are used in reconstruction calculations to generate realistic 3D images and to calculate morphological parameters. A major application of μCT has been to quantify the 3D microstructure of bone and to provide quantitative information about its functionality, porosity, and mineral density, making the technique useful for early detection of various bone pathologies, including osteoporosis.65–68
μCT can also be used to study calcification of the arterial wall68,69 and enables nondestructive visualization and localization of CHA in very thin tissue slices, although it does not allow for delineation of other plaque components (Figure 2). Two morphological distributions are observed: (1) calcification nodules localized mainly in the necrotic core or luminal surface, and (2) calcification plates localized more to the medial layer, often extending around a substantial fraction of its circumference. Calibrating the μCT system with known amounts of CHA is done by scanning a 96-well microtiter plate having wells filled with different amounts of hydrated CHA. The sum of electron dense areas in successive slices of CHA in each well enables construction of a standard calibration curve that can be used for calculation of unknown samples.
Figure 2. μCT images of a CCA. Axial slice images (0.3 mm) reveal a large calcification plate (a and b) and small nodules (c) in the internal carotid but no calcification in the common carotid (d).
Magnetic Resonance Imaging
Ex vivo samples can be imaged by MRI using coils of different geometries. A solenoid coil (30-mm diameter) is convenient for imaging single samples at very high resolution. A phased array coil (6-cm width) is convenient for multiple samples at high resolution. MRI analysis of CEA specimens can be performed using the same sample holder but smaller sample tubes than used for CCA imaging.
Representative magnetic resonance (MR) images of CEA tissues are shown in Figure 1. Plaque components, including collagenous cap, necrotic core, hemorrhage, and calcification can be distinguished in MR images of plaques.70–72 These components can also be mapped according to their contrast weightings, which allow for integration of the components and quantification. Multicontrast-weighted MR images of carotid plaques have also been used to develop classification maps useful in distinguishing plaque components more effectively69 (Table).
In a study of left and right CCA, MRI and EBCT were used to determine arterial wall volume and calcification score.73 The results indicated that total wall volume and plaque calcification burden were similar for the left and right arteries, suggesting that atherosclerosis is a bilaterally symmetrical disease.
In PDW images of CEA tissues, regions of calcification appear dark (Figure 3b). When these tissues are embedded in paraffin, the calcified regions appear white (Figure 3a). Integration of the calcified regions visualized by each method provided 2 sets of integrated areas (MRI and embedded tissue imaging) that were highly correlated (R2=0.99; Figure 3c).
Figure 3. a, A CEA specimen was microdissected into segments 1.0 cm in length. Selected segments were fixed and embedded in paraffin by conventional methods. From each block, excess paraffin was lifted with a microtome until a full artery cross-section was exposed. The fixed tissue cross-sections were digitally photographed and contrast-manually enhanced with the NIH image analysis program ImageJ 1.30v. b, Each digital image was matched using eFilm Workstation 1.8.3 with the MRI image of the closest morphology. For each image, the total artery area, total lumen area, and total calcified area were circumscribed independently using freehand area selection in ImageJ and measured by automated integration. Percent calcification was then calculated for each image. c, Comparison of percent calcification in CEA specimens detected by MRI (b) and embedded tissue imaging (ETI; a; S. Marvel and J. Morrisett, unpublished results, 2005).
Three-D images have been obtained of human CCA using multicontrast-weighted fast spin-echo imaging.74 A cluster analysis technique called spatially enhanced cluster analysis objectively classified and quantified multicontrast MR images. The cluster technique divides data into groups with strong associations by iteratively minimizing a characteristic of the cluster. Using this technique, plaque components such as calcification can be differentiated by color and then quantified.
31P MR Spectroscopy
MR spectroscopy (MRS) is useful for quantifying nuclei in specific magnetic environments and has been used extensively to study biomolecules in isotropic solutions. Magic angle spinning (MAS) extends the power of MRS to include determining chemical and structural properties of anisotropic liquid crystalline and solid samples. The mineral content of bone can be quantified using 31P MRS because the phosphate of CHA is distinguishable from Pi, from phosphorylated metabolites dissolved in the cytosol, and from the polar head groups of phospholipids in membranes.75,76 Accordingly, 31P MAS MRS can be used for rapid quantification of CHA in atherosclerotic plaques.77,78
In a study of lipid phases and CHA deposits in human atherosclerotic lesions, a plaque with low lipid content (weak 13C MAS MRS signals) and extensive calcification (strong 31P MAS MRS signals) has been used to determine CHA content.77 After delipidation of the plaque, the 31P MAS MRS signal intensity showed no change, indicating that the 31P signal resulted from nonlipid phosphorus. Based on the intensity of the 31P MAS MRS peak, the phosphorus content could be calculated, followed by stoichiometric conversion to CHA content.
The integrated 31P signal intensity in plaques can be calibrated using synthetic CHA and chicken bone powder as reference compounds.78 Comparisons of 31P peak intensities showed that the chicken bone powder provided the best calibration, presumably because the synthetic CHA has a more ordered crystalline structure than the biological samples. This technique is somewhat destructive because delipidation leaves the tissue in a non-native state. 31P MAS MRS is generally restricted to ex vivo samples <1 cm3 (Table).
Tissue Digital Photography
Digital photographs of CEA specimens are useful for documenting tissue features lost during processing (eg, thrombus and calcification) for microscopy and can capture subtle textural and morphological features not detected by other techniques (Figure 4). Although differences in photographic color are useful for qualitatively distinguishing between calcified and lipid-rich regions, the contrast is not sufficient to accurately quantify the 2 types of plaque components. However, estimating calcium in these tissues becomes feasible when they are embedded in paraffin and digitally photographed (Figure 3). The total artery area, total lumen area, and total calcified area can be integrated and these areas used to calculate the percent calcification for each image. Although paraffin-thin sections frequently lose some of the calcified component during cutting, the remaining block has a smooth surface that facilitates quantitation (Table).
Figure 4. Digital photograph of a fresh CEA specimen (a) illustrating the presence of extensive calcification and thrombus in segment I3 (b). Thrombus appears as a dark red area, and calcification appears as a white–pink area. The shiny white areas are optical artifacts.
Histology
Conventional histology is used widely to analyze the structure of animal and plant tissues. Fixed and stained CEA specimens viewed by light microscopy are useful for identifying plaque components.79 For calcified tissues in paraffin blocks, cutting intact cross-sections can be difficult. Exemplary images from histological analysis are shown in Figure 5. Cutting calcification in frozen sections is even more challenging, usually requiring at least treatment with nitric acid or EDTA. However, these treatments cause calcium depletion and can reduce immunoreactivity of calcium-binding proteins. The percentages of calcification and other plaque components can be determined by area integration.80,81
Figure 5. From the internal branch of the left carotid artery, fibro-fatty lesions have been segmented and fixed in paraffin blocks. Then 10-μm histology sections prepared from original blocks were stained with: a, Oil Red O, to stain lipid purple, and nuclei brown; b, Van Kossa, to stain calcium salts black, nuclei red, and cytoplasm light pink; and c, trichrome, to stain muscle fibers red, collagen blue, and nuclei blue to black. These sections illustrate distribution of small calcification nodules through the midintimal area (heavy arrow) and loss of calcification from a deep-intimal area near 2:00 (light arrow).
Embedding tissue in plastic helps prevent loss of calcification during sectioning while retaining stain and antibody reactivity. Representative plastic embedded thin sections are shown in Figure 6. Paraffin blocks and frozen sections are attended by significant loss of calcification; however, the time-consuming process of tissue embedding in plastic allows retention of calcification and its quantification (Table).
Figure 6. X-ray image and corresponding thin-section light micrographs from right CCA fixed in 10% formalin. After fixation, the CEA samples are dehydrated in a graded series of ethanol washes and embedded in glycomethylmethacrylate. After polymerization, thin sections (10 to 30 um) are prepared using the Exakt System modified sawing microtome technique.84 Sections are stained with a Modified Goldner Tri-Chrome reagent for plastic sections for which calcification appears blue–green.
Chemical Analysis
Atomic absorption spectroscopy (AAS) is a sensitive analytical technique used to determine the concentration of metals in liquid samples. In their elemental form, metals absorb ultraviolet light when excited by heat, and each metal has a characteristic wavelength that will be absorbed. The amount of light absorbed is proportional to the concentration of the element in the solution. Measurements are made separately for each element of interest. The method is very sensitive and can measure trace elements down to the parts per million level. Because calcium is one of the metals detectable by AAS, this method is useful in determining calcium concentration in CEA samples. Stoichiometry can be used to calculate the quantity of CHA in the samples (Table).
Another method for chemically analyzing CHA in CEA samples uses the phosphomolybdate reagent, which quantifies the amount of elemental phosphorus in a sample.82 The sample is digested in sulfuric acid and oxidized with hydrogen peroxide to liberate elemental phosphorus. Ammonium molybdate and 1-amino-2-naphthol-4-sulfonic acid are added to produce a solution that absorbs at 830 nm. The concentration of phosphorus is determined from a calibration curve of various concentrations of a standard phosphate salt. The stoichiometry of CHA, Ca10(PO4)6(OH)2, can be used to convert the measured phosphorus concentration to CHA mass. A useful alternative method uses o-cresolphthalein for calcium determination.83 Fura and Indo ratiometric calcium indicators are excited with UV light, allowing for quantification of calcium content as well.84
Concluding Comments
Selecting an appropriate method of calcification quantification depends on the nature of the specimen and the desired results (Table). Although the in vivo methods of US, EBCT, and MRI are all capable of differentiating plaque components, IVUS and MRI have the highest spatial resolution. In ex vivo analyses, μCT and MRI are the principal methods for visualizing calcified thin slices and creating 3D reconstruction of the specimen. Digital photography is useful for capturing a visual record of intact tissues and tissue fragments before processing by embedding or homogenization. Histological sections provide morphological information that is currently the gold reference standard for validating many imaging techniques. 31P MAS MRS and the microphosphorus chemical assay for calcification quantification rely on chemical stoichiometry to convert the concentration of phosphorus atoms to CHA. The chemical assay is more amenable to high throughput but requires sample destruction. AAS is probably the most sensitive method for calcium quantification; it has the same capabilities and limitations as those of the microphosphorus assay. These qualitative and quantitative methods will likely see increasing use in future investigations of vascular calcification.
Acknowledgments
This work was supported in part by the National Institutes of Health grants HL63090 and HL07812. The authors thank Catherine Ambrose, PhD, and Tiffany Sheffield of the Bone Histomorphometry and Biomaterials Laboratory in the Department of Orthopaedic Surgery at the University of Texas-Houston Medical School for the plastic embedded histochemical preparations.
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