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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 DI 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. (Ant J Pathol 2011, 178:2879-2887; DOI: 10.1016/j.ajpath.2011.02.004)

BackgroundVascular 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 StudyThis 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 ApproachTo 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.ResultsFrom 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.ConclusionOur 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.

Human coronary arteries in both early and advanced stages of atherosclerosis were studied for understanding the nature of Ca deposits in terms of their Ca/P mass ratio with the use of Eindhoven nuclear microprobe. The ratios observed differed appreciably from that of hydroxyapatite (2.15), which is commonly referred to as the Ca component in arteries affected by atherosclerosis. Very small granular calcified structures were found in an early stage. Broadly, two types of calcified structures were observed: the Ca/P ratio corresponding to the advanced stage (3.18) was found higher compared to those for the early stage (1.46–1.77). It has been argued that besides phosphates, the large Ca/P ratio corresponding to intense calcification may be due to a Ca deposit with a different anion (oxalate, hydroxide or even carbonate), while, the lower ratios may be due to an additional inorganic phosphate. The distribution of S/P ratios was used to account for enhanced deposition of lipid materials in the affected area at the expense of proteins.

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.

Principal component analysis was applied to identify normal and pathological structures using combinations of levels of phosphorus, sulphur, chlorine, potassium and calcium in early stages of atherosclerotic coronary arteries. An inverse correlation was found between the combination of Ca–P, and that of S–Cl–K. This finding points towards the presence of smaller calcified deposits in an early phase of atherosclerosis in the human coronary artery. A possible location of cholesterol or some other carbon-based materials could also be identified. Further, a histogram method proved to be useful for characterising calcified structures of Ca/P mass ratio lower than that of hydroxyapatite. An empirical formula of (1− n )Ca 3 (PO 4 ) 2 · n A m (PO 4 ) is suggested to explain composition of these calcified structures.