Intra-Section Analysis of Human Coronary Arteries Reveals a Potential Role for Micro-Calcifications in Macrophage Recruitment in the Early Stage of Atherosclerosis.

BACKGROUND: Vascular 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. AIM OF THIS STUDY: This 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. TECHNICAL APPROACH: To 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. RESULTS: From 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. CONCLUSION: Our 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.

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.

Microcalcifications in early intimal lesions of atherosclerotic human coronary arteries.

Although calcium (Ca) precipitation may play a pathogenic role in atherosclerosis, information on temporal patterns of microcalcifications in human coronary arteries, their relation to expression of calcification-regulating proteins, and colocalization with iron (Fe) and zinc (Zn) is scarce. Human coronary arteries were analyzed post mortem with a proton microprobe for element concentrations and stained (immuno)histochemically for morphological and calcification-regulating proteins. Microcalcifications were occasionally observed in preatheroma type I atherosclerotic intimal lesions. Their abundance increased in type II, III, and IV lesions. Moreover, their appearance preceded increased expression of calcification-regulating proteins, such as osteocalcin and bone morphogenetic protein-2. In contrast, their presence coincided with increased expression of uncarboxylated matrix Gla protein (MGP), whereas the content of carboxylated MGP was increased in type III and IV lesions, indicating delayed posttranslational conversion of biologically inactive into active MGP. Ca/phosphorus ratios of the microcalcifications varied from 1.6 to 3.0, including amorphous Ca phosphates. Approximately 75% of microcalcifications colocalized with the accumulation of Fe and Zn. We conclude that Ca microprecipitation occurs in the early stages of atherosclerosis, inferring a pathogenic role in the sequel of events, resulting in overt atherosclerotic lesions. Microcalcifications may be caused by local events triggering the precipitation of Ca rather than by increased expression of calcification-regulating proteins. The high degree of colocalization with Fe and Zn suggests a mutual relationship between these trace elements and early deposition of Ca salts.

Microcalcifications in atherosclerotic lesion of apolipoprotein E-deficient mouse.

Evidence is accumulating that calcium-rich microdeposits in the vascular wall might play a crucial role in the onset and progression of atherosclerosis. Here we investigated an atherosclerotic lesion of the carotid artery in an established murine model, i.e. the apolipoprotein E-deficient (APOE(-/-) ) mouse to identify (i) the presence of microcalcifications, if any, (ii) the elemental composition of microcalcifications with special reference to calcium/phosphorus mass ratio and (iii) co-localization of increased concentrations of iron and zinc with microcalcifications. Atherosclerosis was induced by a flow-divider placed around the carotid artery resulting in low and high shear-stress regions. Element composition was assessed with a proton microprobe. Microcalcifications, predominantly present in the thickened intima of the low shear-stress region, were surrounded by areas with normal calcium levels, indicating that calcium-precipitation is a local event. The diameter of intimal microcalcifications varied from 6 to 70 µm. Calcium/phosphorus ratios of microcalcifications varied from 0.3 to 4.8, mainly corresponding to the ratio of amorphous calcium-phosphate. Increased iron and zinc concentrations commonly co-localized with microcalcifications. Our findings indicate that the atherosclerotic process in the murine carotid artery is associated with locally accumulated calcium, iron and zinc. The calcium-rich deposits resemble amorphous calcium phosphate rather than pure hydroxyapatite. We propose that the APOE(-/-) mouse, in which atherosclerosis was evoked by a flow-divider, offers a useful model to investigate the pathophysiological significance of accumulation of elements such as calcium, iron and zinc.

Early calcifications in human coronary arteries as determined with a proton microprobe.

The current paradigm reads that calcifications characterize the advanced and complex lesions in the atherosclerotic process. To explore the possibility that coronary artery wall calcifications already commence at an early stage of atherosclerosis, a combination of proton beam techniques with a (sub-) micrometer resolution, i.e., micro-proton induced X-ray emission, backward and forward scattering spectroscopy, was applied on human coronary arteries with lesions preceding overt atheromas. The detection limits of phosphorus and calcium in each separate pixel, 0.88*0.88 microm2 in size, were approximately 150 and 80 microg/g dry weight, respectively. Calcium distributions of entire coronary artery cross section were obtained, and calcifications were demonstrated at a preatheroma stage of the atherosclerotic process. The size of the microcalcifications varied between 1 and 10 microm. The composition of the microcalcifications was deduced from the calcium-to-phosphorus ratio. In order to quantify this ratio, the thickness of the specific X-ray absorber used for PIXE had to be accurately determined. Also, thick target PIXE calculations were performed and the method was validated. The calcium-to-phosphorus ratios of the microcalcifications were assessed with good accuracy and varied from 1.62 to 2.79, which corresponds with amorphous calcium phosphate.