Coronary Artery Calcium Scores: Current Thinking and Clinical Applications

Most incident coronary disease occurs in previously asymptomatic individuals who were considered to be at a lower risk by traditional screening methods. There is a definite advantage if these individuals could be reclassified into a higher risk category, thereby impacting disease outcomes favorably. Coronary artery calcium scores have been recognized as an independent marker for adverse prognosis in coronary disease. Multiple population based studies have acknowledged the shortcomings of risk prediction models such as the Framingham risk score or the Procam score. The science behind coronary calcium is discussed briefly followed by a review of current thinking on calcium scores. An attempt has been made to summarize the appropriate indications and use of calcium scores

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Element analysis with a proton microprobe of early atherosclerotic lesions

Atherosclerosis is a progressive inflammatory vascular disease accompanied by a gradual build-up of cholesterol in the artery walls. The associated chronic inflammatory process leads to tissue damage in the vascular wall as a consequence of an excessive inflammatory response. Large calcified deposits of the intimal lesions are a prominent feature of advanced atherosclerotic lesions. The degree of these calcifications in the coronary arteries correlates highly with the severity of coronary plaque burden and is thus associated with increased cardiovascular risk. In lesions preceding atheromas calcifications are rarely observed, most likely because of insufficient sensitivity and/or specificity of the techniques used. It is generally thought that calcifications are composed of hydroxyapatite in advanced atherosclerotic lesions and that atherosclerotic calcification occurs passively as the result of precipitation of calcified material in cores of dying cells. However, recent findings might indicate that atherosclerotic calcification is an actively regulated process involving, among others, bone matrix regulatory proteins. Most information on atherosclerotic calcifications deals with advanced intimal lesions and chemical analysis was mainly performed on larger, solid crystals. Data about the distribution and composition of the calcifications at a very early stage of the atherosclerotic process are scarce, as is information whether trace-elements could be involved in the calcification process. Chemical bulk analysis of the mineral part of human aortas, with advanced atherosclerotic lesions, showed that the average mass ratio of calcium to phosphorus is close to the ratio of hydroxyapatite, i.e. 2.16, which is the main constituent of bone tissue1. Though, others found lower ratios of calcium to phosphorus in atherosclerotic plaques2, 3. Also traces of iron and zinc were occasionally observed in these bulk analyses. In a pioneering study of by Pallon et al.4 the co-localization of iron and zinc with calcium granules, 10 to 20 µm in diameter, was observed in early atherosclerotic lesions in human coronary arteries. These observations incited our interest in the pathological significance of calcium precipitations in the early phase of atherosclerosis and the role of trace-elements, such as iron and zinc. The pathophysiological background of the subject of this thesis is summarized in chapter 2. To investigate the distribution, composition (calcium-to-phosphorus mass ratio) and trace-element contents of calcifications in early atherosclerotic lesions a technique was applied with high sensitivity, specificity and resolution, i.e., micro-Proton Induced X-ray Emission in combination with Backscattering and Forward Scattering Spectroscopy5, 6. With this technique pixel wise element concentration maps can be created to visualize the distribution of elements, like calcium and phosphorus, in cryosections of artery wall tissues. The technique is very sensitive enabling element concentration maps of trace-elements like iron and zinc to be obtained. For this technique high energy protons are required and these are produced utilizing an acquired accelerator (SingletronTM, High Voltage Engineering Europe B.V., Amersfoort, the Netherlands). The accelerator is connected to a proton microprobe setup, which was optimized for the detection of trace-elements at hotspots of calcium. The proton-beam was miniaturization, as the size of the calcifications is expected to be in the range of micrometers or even smaller. The settings for the object and aperture diaphragms were optimized as well as for the quadrupoles. The profiles and sizes of the proton beam were measured. The measured beam size has been reduced to about 0.5 to 0.5 µm2 at best. The position of the beam relative to the position of the sample and its size were constant during the time needed for a measurement. A beryllium absorber with calibrated thickness was placed in front of an X-ray detector for the detection of characteristic X-rays (PIXE) corresponding to, for instance, phosphorus and calcium. A second ultra-LEGe detector with a larger surface area was introduced for the detection of X-rays (PIXE) corresponding to iron and zinc. The solid angles of the detectors were optimized for the detection of phosphorus, calcium and trace-elements. The detection limits were in the order of µg/g for the trace-elements. A new data acquisition hardware and software system with graphical user interface was introduced which remotely controlled the proton microprobe setup. This made it possible to acquire large datasets. New analysis software with graphical user interface was written, which had the advantage of monitoring the experiment in a flexible way and data analysis speed was improved dramatically (chapter 3). The proton microprobe was applied to obtain directly the distribution of calcifications in entire cross-sections of human coronary arteries with lesions preceding artheromas. This objective required that consecutive scans were made that cover the whole cross section of a coronary artery wall to obtain an overview of the calcium distribution in the entire artery wall. The composition of the micro-calcifications was deduced from the calcium-to-phosphorus ratio. In order to quantify the calcium-to-phosphorus ratio a specific X-ray absorber used for PIXE was gauged with a reference sample. Inside the sample, protons can lose their energy and X-rays can be attenuated. These two effects influence the estimated values of the calcium-to-phosphorus ratio and, hence, corrective calculations were validated. To obtain the calcium-to-phosphorus ratio of a calcification the calcium and phosphorus concentrations of pixels belonging to the micro-calcification were represented in a scatter plot. The slope of a linear fit through the calcium and phosphorus concentrations in the scatter plot provides a reliable value of the calcium-to-phosphorus mass ratio of the micro-calcification (chapter 4). Tissue sections of coronary artery walls containing AHA (American Heart Association) type I to VII atherosclerotic lesions from patients who died from non-cardiac causes were the subject of investigation. Calcium deposits at a (sub)micrometer scale were observed in the thickened intima already in early pre-atheromatous stages of atherosclerosis (type II lesions), predominantly in the abluminal region of the intima. The occurrence of micro-calcifications increased in more severe stages of atherosclerosis. In type VII lesions big lumps of calcified material could be easily observed. The composition of the micro-calcifications, i.e. the calcium-to-phosphorus mass ratio, was investigated in type II, III, and IV lesions and micro-calcifications displayed a range of values (1.62 to 2.99). However, the calcium-to-phosphorus ratio was very homogeneous within a given hotspot: each micro-calcification area displayed a unique calcium-to-phosphorus ratio, which might be dependant on the local environment. The range of calcium-to-phosphorus mass ratios strongly suggests that the micro-calcifications consisted of amorphous calcium-phosphate crystals and was not restricted to hydroxyapatite. However, it can not be excluded that the hotspots consist of a mixture distinct calcium-phosphate compounds. For all the calcium hotspots described above also trace-elements were monitored. The detection limits for iron, copper and zinc were respectively 7, 5 and 5 µg/g. In none of the areas investigated copper could be found above the detection limit. The majority of the calcifications showed co-localisation with hotspots of iron and zinc. This finding might imply a pathogenic relationship between calcium precipitation on the one hand and iron and zinc on the other in these early atherosclerotic lesions in human coronary arteries. The pathophysiological significance and the underlying mechanism of this relationship are, however, incompletely understood. The involvement of bone matrix regulatory proteins during the calcification process was studied with immunohistochemistry. The occurrence of micro-calcifications preceded the expression of proteins actively involved in bone formation such as Matrix Gla Protein (MGP). Because MGP possesses the property to bind calcium ions and, hence, to inhibit the formation of insoluble calcium salts7, 8, the increased expression of MGP following the formation of micro-calcifications could point to an adaptive mechanism in the affected intima to mitigate or prevent further deposition and/or growth of calcified material. Increased expression of Bone morphogenetic protein-2 and Osteocalcin could only be observed in advanced stages of atherosclerosis when an atheroma had formed. Osteocalcin (OC or bone Gla protein) also exerts a mitigating effect on bone formation, but Bone Morphogenetic Proteins (BMP) are known to mediate ectopic bone formation. The same holds for Core binding factor-a1, showing a diffuse expression in advanced atherosclerotic lesions. This protein is known to regulate osteoblast and chondrocyte differentiation. The present findings infer that the formation of calcium phosphate granules is an early event in the atherosclerotic process and that the early deposition of calcium-rich material at the microscale is most likely not regulated by bone matrix regulatory proteins (chapter 5). In a carotid artery of an ApoE-/- mouse with an advanced atherosclerotic lesion (AHA type V lesion) also showed the presence of micro-calcifications. Atherosclerosis occurred in the low shear stress region. The high shear stress region remained unaffected. The calcifications were present both in the thickened intima and in the media of the low shear stress region. The calcium-to-phosphorus mass ratios ranged from 0.65 to 5.9, which is a much broader range than observed in human coronary arteries. These findings indicate that the composition differs between human and murine atherosclerotic calcifications. The question whether this implies that the nature of the atherosclerotic process is different between human and murine vascular tissue warrants further investigations (chapter 6). The findings described in this thesis are summarized and discussed in chapter 7. The main conclusions of the studies described in this thesis are that calcifications are present in very early atherosclerotic lesions as measured with a combination of proton-beam techniques. The composition of the micro-calcifications, i.e., the calcium-to-phosphorus mass ratio, in human coronary arteries displayed a range of values (1.62 to 2.99), strongly suggesting that the micro-calcifications consisted of amorphous calcium-phosphate crystals. The calcifications showed co-localisation with iron and zinc. This finding might imply a pathogenic relationship between calcium precipitation on the one hand and iron and zinc on the other. The present findings also indicate that the early deposition of calcium-rich material precedes the expression of bone matrix regulatory proteins

High-resolution 3D X-ray imaging of intracranial nitinol stents

Introduction To assess an optimized 3D imaging protocol for intracranial nitinol stents in 3D C-arm flat detector imaging. For this purpose, an image quality simulation and an in vitro study was carried out. Methods Nitinol stents of various brands were placed inside an anthropomorphic head phantom, using iodine contrast. Experiments with objects were preceded by image quality and dose simulations. We varied X-ray imaging parameters in a commercially interventional X-ray system to set 3D image quality in the contrast–noise–sharpness space. Beam quality was varied to evaluate contrast of the stents while keeping absorbed dose below recommended values. Two detector formats were used, paired with an appropriate pixel size and X-ray focus size. Zoomed reconstructions were carried out and snapshot images acquired. High contrast spatial resolution was assessed with a CT phantom. Results We found an optimal protocol for imaging intracranial nitinol stents. Contrast resolution was optimized for nickel–titanium-containing stents. A high spatial resolution larger than 2.1 lp/mm allows struts to be visualized. We obtained images of stents of various brands and a representative set of images is shown. Independent of the make, struts can be imaged with virtually continuous strokes. Measured absorbed doses are shown to be lower than 50 mGy Computed Tomography Dose Index (CTDI). Conclusion By balancing the modulation transfer of the imaging components and tuning the high-contrast imaging capabilities, we have shown that thin nitinol stent wires can be reconstructed with high contrast-to-noise ratio and good detail, while keeping radiation doses within recommended values. Experimental results compare well with imaging simulations

Intra-section analysis of human coronary arteries reveals a potential role for micro- calcifications in macrophage recruitment in the early stage of atherosclerosis

Background\u3cbr/\u3e\u3cbr/\u3eVascular calcification is associated with poor cardiovascular outcome. Histochemical analysis of calcification and the expression of proteins involved in mineralization are usually based on whole section analysis, thereby often ignoring regional differences in atherosclerotic lesions. At present, limited information is available about factors involved in the initiation and progression of atherosclerosis.\u3cbr/\u3e\u3cbr/\u3eAim of This Study\u3cbr/\u3e\u3cbr/\u3eThis study investigates the intra-section association of micro-calcifications with markers for atherosclerosis in randomly chosen section areas of human coronary arteries. Moreover, the possible causal relationship between calcifying vascular smooth muscle cells and inflammation was explored in vitro.\u3cbr/\u3e\u3cbr/\u3eTechnical Approach\u3cbr/\u3e\u3cbr/\u3eTo gain insights into the pathogenesis of atherosclerosis, we performed analysis of the distribution of micro-calcifications using a 3-MeV proton microbeam. Additionally, we performed systematic analyses of 30 to 40 regions of 12 coronary sections obtained from 6 patients including histology and immuno-histochemistry. Section areas were classified according to CD68 positivity. In vitro experiments using human vascular smooth muscle cells (hVSMCs) were performed to evaluate causal relationships between calcification and inflammation.\u3cbr/\u3e\u3cbr/\u3eResults\u3cbr/\u3e\u3cbr/\u3eFrom each section multiple areas were randomly chosen and subsequently analyzed. Depositions of calcium crystals at the micrometer scale were already observed in areas with early pre-atheroma type I lesions. Micro-calcifications were initiated at the elastica interna concomitantly with upregulation of the uncarboxylated form of matrix Gla-protein (ucMGP). Both the amount of calcium crystals and ucMGP staining increased from type I to IV atherosclerotic lesions. Osteochondrogenic markers BMP-2 and osteocalcin were only significantly increased in type IV atheroma lesions, and at this stage correlated with the degree of calcification. From atheroma area type III onwards a considerable number of CD68 positive cells were observed in combination with calcification, suggesting a pro-inflammatory effect of micro-calcifications. In vitro, invasion assays revealed chemoattractant properties of cell-culture medium of calcifying vascular smooth muscle cells towards THP-1 cells, which implies pro-inflammatory effect of calcium deposits. Additionally, calcifying hVSMCs revealed a pro-inflammatory profile as compared to non-calcifying hVSMCs.\u3cbr/\u3e\u3cbr/\u3eConclusion\u3cbr/\u3e\u3cbr/\u3eOur data indicate that calcification of VSMCs is one of the earliest events in the genesis of atherosclerosis, which strongly correlates with ucMGP staining. Our findings suggest that loss of calcification inhibitors and/or failure of inhibitory capacity is causative for the early precipitation of calcium, with concomitant increased inflammation followed by osteochondrogenic transdifferentiation of VSMCs.\u3cbr/\u3

Small calcified coronary atherosclerotic plaque simulation model:minimal size and attenuation detectable by 64-MDCT and MicroCT

Zero calcium score may not reflect the absence of calcifications as small calcifications could be missed. This study aimed to evaluate minimal size and minimal attenuation of coronary calcifications detectable by computed tomography (CT) and to determine the minimal spatial resolution required for detecting calcification onset. Using open source CT simulation software, CTSim(©), several 50%-stenotic coronary artery phantoms were designed with 5 μm resolution, realistic morphology and tissue-specific Hounsfield Unit (HU) values. The plaque had an attenuation resembling fibrous plaque and contained a single calcification. X-ray projections were simulated with settings resembling non-contrast-enhanced 64 multi detector-row CT (64-MDCT). Scanning and reconstruction were simulated with spatial resolution of a 64-MDCT (0.4mm) and of a MicroCT (48 μm). Starting from a single calcium granule, the calcification was simulated to grow in size and attenuation until it could be detected using clinically accepted calcium determination scheme on MicroCT and 64-MDCT images. The smallest coronary calcifications detectable at MicroCT and 64-MDCT, which had a realistic attenuation (−1,024 to 3,072 HU), were of 25 μm and 215 μm diameter, respectively. The area was overestimated 7.7 and 8.8 times, respectively. Calcifications with smaller size need to have an unrealistically high attenuation to be detectable by 64-MDCT. In conclusion, 64-MDCT is only able to detect coronary calcifications with minimal diameter of 215 μm. Consequently, early onset of calcification in coronary plaque will remain invisible when using CT and a zero calcium score can not exclude the presence of coronary calcification