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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2010 Dec;91(6):485–494. doi: 10.1111/j.1365-2613.2010.00729.x

Microcalcifications in atherosclerotic lesion of apolipoprotein E-deficient mouse

Nicola Debernardi *, Ruben B Roijers *, Rob Krams †, Rini de Crom ‡, Peter HA Mutsaers *, Ger J van der Vusse §
PMCID: PMC3010546  PMID: 20804542

Abstract

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.

Keywords: atherosclerosis, iron, microcalcification, proton microprobe, zinc

Atherosclerotic lesions are generally observed in the inner curvature of curved arterial segments, where low shear stress prevails, and near side-branches, where oscillatory shear stress prevails (Caro et al. 1969; VanderLaan et al. 2004). In addition to accumulation of lipid-rich material, deposition of calcium-phosphate salts is a common feature in advanced atherosclerotic lesions (Lusis 2000; Libby 2001; Proudfoot & Shanahan 2001; Budoff & Gul 2008). Evidence is accumulating that precipitation of insoluble calcium salts at a (sub)micrometre scale, so-called microcalcifications (Roijers et al. 2008), in the affected wall is an early phenomenon in the sequel of events eventually resulting in overt plaque formation (Tanimura et al. 1983; Bobryshev et al. 1995; Pallon et al. 1995; Stary 2001). It has been suggested that local accumulation of trace elements such as iron and zinc might also play a role in the onset of atherosclerosis (Heinecke 2003; Minqin et al. 2003; Stadler et al. 2004; Lapenna et al. 2007).

Genetically engineered mice models are currently applied to elucidate the mechanisms underlying the onset and progression of atherosclerotic lesions (Carmeliet et al. 1998; Hofker et al. 1998). However, the application of these models in investigating the arterial deposition of microcalcifications in atherosclerosis-prone experimental animals is limited. A recent study of Clarke et al. (2008) revealed that in apolipoprotein E-deficient (APOE−/−) mice, a chronic low level of vascular smooth muscle cell apoptosis evokes local precipitation of calcium-rich material. In earlier investigations on APOE−/− mice, Rattazzi et al. (2005) claimed that the small deposits of calcium-rich material were composed of hydroxyapatite, being the main component of calcified bone tissue. Despite indications that the APOE−/− mouse model could serve as a powerful experimental tool to reveal crucial steps in the overall atherosclerotic process, information on the chemical composition of microcalcifications and co-localization with elements such as iron and zinc is virtually lacking.

The main aims of the present, explorative study were to investigate (i) the presence of microcalcifications in an atherosclerotic lesion in the APOE−/− mouse carotid artery, (ii) the elemental composition of the microcalcifications, if any, with special reference to the calcium/phosphorus mass ratio and (iii) the co-localization of microcalcifications and increased concentrations of iron and zinc.

The atherosclerotic lesion was induced by chronic exposure of the vessel wall to low shear stress by a flow divider (Cheng et al. 2006). The calcium/phosphorus mass ratios were determined to explore whether calcium-rich micro-deposits mimic the composition of bone material. Accumulation of iron and zinc in the lesion was measured to assess their potential role of these trace elements in the atherosclerotic process. The element concentrations were assessed with a proton microprobe technique, details of which were published recently (Roijers et al. 2008).

Materials and methods

Experimental animals

APOE−/− mice (males, 15–20 weeks old) in C57BL/6J background were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). During the experimental period animals were fed a Western type diet containing 15% (w/v) cacao butter and 0.25% (w/v) cholesterol (diet W, Hope Farms, Woerden, the Netherlands). All experiments were performed in compliance with institutional (Erasmus MC, Rotterdam, the Netherlands) and international guidelines.

Induction of atherosclerosis by low shear stress

To induce standardized changes in shear stress, a shear stress modifier was manufactured of thermoplastic polyether-keton. The cast consists of two longitudinal halves of a cylinder with a cone shaped lumen. The geometry of the cast has been designed with computational flow dynamics software to produce vortices downstream of the cast when placed around the common carotid artery. The upstream inner diameter is 500 μm (non-constrictive) and gradually decreases to 250 μm at the down stream side of the cast (constrictive). This tapering induces a gradual increase of shear stress (high shear stress region). In addition, the constrictive stenosis decreases the blood flow, resulting in a low shear stress region upstream from the cast (Cheng et al. 2006).

After 2 weeks of Western diet, the animals were anaesthetized with isoflurane, and the anterior cervical triangles were accessed by a sagittal anterior neck incision. The right common carotid artery was dissected from circumferential connective tissues. The cast was placed around the right common carotid artery, tissue was closed and the animals were allowed to recover. Animals with cast implants were sacrificed at 9 weeks after surgery, implying that the animals received the Western diet for 11 weeks.

Tissue preparation and histology

From a group of more than 40 animals, 1 mouse was randomly selected for detailed element analysis of the affected carotid vessel wall. The other animals were used in a separate study (Cheng et al. 2007).

The animal was anaesthetized with isoflurane and subjected to perfusion fixation as described before (Cheng et al. 2006, 2007). The entire aortic arch with the origins of the right and the left common carotid arteries including 5 mm of these vessels was carefully dissected, embedded in Tissue Tek and frozen in liquid nitrogen. Cryosections were cut at a thickness of about 10 μm in a cryostat microtome (−30 °C) using Teflon-coated disposable knifes and subsequently collected on Pioloform films (thickness 100 ± 10 nm) (Agar Scientific Ltd, Stansted, UK) for PIXE analysis. Both knife and Pioloform films were precooled in the cryostat. The sections on Pioloform were dried in the cryo-microtome for at least 2 h. Adjacent sections were collected on glass slides. Lesion morphology was evaluated on haematoxylin-and-eosin-stained (H&E) sections. One tissue section from the high and one from the low shear stress region were selected at random for further analysis of element composition.

Instrumentation and analysis

The element composition of the tissue sections was assessed with proton microprobe techniques. Measurements were performed at the Eindhoven University of Technology with a 3-MeV proton beam accelerated with a 3.5-MV Singletron™ (High Voltage Engineering Europe B.V., Amersfoort, the Netherlands). The Singletron™ is connected to a proton microprobe setup. Details of the microprobe setup can be found elsewhere (Mutsaers 1996). Element analyses were carried out using Proton Induced X-ray Emission (PIXE) in combination with Backscattering Spectroscopy and Forward Scattering Spectroscopy. The PIXE technique is used to determine the content (g/cm2) of all minor and trace elements present in the tissue section with atomic numbers higher than 11; Backscattering Spectroscopy is used to determine the tissue content of the major elements, i.e. carbon (C), oxygen (O) and nitrogen (N), and Forward Scattering Spectroscopy is used to determine the hydrogen (H) content. The combination of the three techniques results in the concentration (μg/g dry weight of tissue) of the elements of interest, such as sulphur (S), phosphorus (P), calcium (Ca), iron (Fe) and zinc (Zn), in addition to chlorine (Cl) and potassium (K), present in the sample (Watt 1996).

A relatively short measurement was performed on a sample to determine the hydrogen and calcium yield distributions (i.e. the distribution of the number of counts per position, not corrected for the thickness of the sample). These distributions were used for comparison with the H&E stained pictures and subsequently to localize and identify areas of high Ca yield. In the next step, an area of interest was chosen and measurements with higher spatial resolution were performed. A previously described method (Roijers et al. 2008) was applied for thickness and elemental composition determination. This method also includes correction for thick target effects. If necessary, the procedure was repeated for a new or smaller area of interest.

Results

Localization of microcalcifications in the atherosclerotic lesion

H&E staining of the low and high shear stress region of the carotid artery of the APOE−/− mouse showed substantial thickening of the intimal layer in the low shear stress region (Figure 1a, area indicated with I). In contrast, in the high shear stress region the vessel wall was virtually unaffected (Figure 1b).

Figure 1.

Figure 1

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H&E stained sections of carotid artery from the low (1) and high (1) shear stress region. I, M and A refer to intima, media and adventitia, respectively. L refers to lumen of carotid artery. Yellow line indicates boundary between intima and media in low shear stress region; green line indicates boundary between media and adventitia.

The intimal lesion in the low shear stress region showed a thick fibrous cap. Previous studies of our research team have shown that in this murine model of atherosclerosis the intimal plaque is rich in macrophages and lipids and poor in vascular smooth muscle cells and collagens (Cheng et al. 2007). The lumen (L) was significantly narrowed when compared to the lumen in the high shear stress region (Figure 1b). No substantial differences could be observed on the H&E staining between high and low shear stress regions with respect to medial (M) and adventitial (A) layers.

Analysis with the proton microprobe revealed that also the hydrogen yield distribution pattern showed the thickened intima and narrowed lumen of the carotid vessel in the low shear stress region as compared to the high shear stress region (Figure 2a and c, respectively).

Figure 2.

Figure 2

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Hydrogen yield distribution in a tissue section from the low (a) and the high (c) shear stress region of mouse carotid artery. The corresponding calcium yield distributions are shown in (b) and (d), respectively. To obtain yield distributions, an adjacent section of the H&E stained section (Figure 1) was analysed with the proton microprobe. I, M and A refer to intima, media and adventitia, respectively. L indicates lumen of the carotid artery. Yield is colour coded from white, blue, red to yellow in ascending order. (Detailed analysis of the areas marked with Y and X is shown in Figures 3 and 4, respectively).

The calcium yield distribution obtained with the proton microprobe revealed calcium hotspots in the thickened intima and occasionally in the medial layer of the low shear stress region (Figure 2b). In contrast, virtually no calcium hotspots could be observed in the high shear stress region (Figure 2d).

The microcalcifications were not homogenously distributed across the thickened intima; they showed a propensity to cluster in a region neighbouring the narrowed lumen (Figure 2a and b, and adjacent H&E stained section shown in Figure 1a).

Element analysis of microcalcifications and their surrounding

With the calcium yield distribution findings as starting point, a number of microcalcifications were selected at random for further element analysis.

First, a detailed concentration distribution scan was made from the area marked with Y in Figure 2b to analyse a number of calcium hotspots observed in the calcium yield distribution of the medial layer.

The concentration distribution scans (Figure 3a–c) show that the medial layer (M) is relatively rich in sulphur (Figure 3a) when compared to the thickened intimal layer (I) and adventitia (A). Absolute values are given in Table 1. The concentration of the other elements measured was not significantly different between adventitia and media in the high shear stress region (Table 1). The same holds for the media, adventitia and thickened intima in the low shear stress region, when elements were measured in areas devoid of microcalcifications.

Figure 3.

Figure 3

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Element concentration distributions, obtained with the proton microprobe, of area marked with Y in Figure 2b of the atherosclerotic lesion in the low shear stress region. Values at the right hand side of the colour bars refer to concentration (μg/g dry weight of tissue) of sulphur (S, a), carbon (C, b) and calcium (Ca, c). I, M and A refer to intima, media and adventitia, respectively. The dimension of the scan is 102 × 102 μm2; beam size is 0.8 × 0.8 μm2 with an average current of 367 pA and a cumulative charge of 26 μC per scan. Pixel size amounts to 3.2 × 3.2 μm2.

Table 1.

Average concentration of elements in the high and low shear stress regions of mouse carotid artery (μg/g dry weight of tissue, except for the last row where the numbers are unitless). The values refer to the mean and standard deviation. The number of data points is on the order of 500 per element. In the low shear stress region, only data from areas devoid of microcalcifications were included in the table

High shear stress tissue Low shear stress tissue
Element Adventitia Media Adventitia Media Intima
C (87 ± 3) × 104 (86 ± 2) × 104 (87 ± 5) × 104 (93 ± 2) × 104 (94 ± 2) × 104
N (8 ± 2) × 104 (9 ± 1) × 104 (8 ± 4) × 104 (3 ± 1) × 104 (2 ± 1) × 104
O (3 ± 1) × 104 (22 ± 8) × 103 (3 ± 2) × 104 (10 ± 4) × 103 (8 ± 4) × 103
P (5 ± 2) × 103 (6 ± 2) × 103 (6 ± 2) × 103 (7 ± 2) × 103 (7 ± 2) × 103
S (4 ± 1) × 103 (60 ± 9) × 102 (40 ± 9) × 102 (60 ± 6) × 102 (4 ± 2) × 103
Cl (34 ± 8) × 102 (39 ± 3) × 102 (27 ± 5) × 102 (20 ± 2) × 102 (16 ± 6) × 102
K (16 ± 4) × 102 (18 ± 3) × 102 (17 ± 3) × 102 (155 ± 8) × 101 (17 ± 6) × 102
Ca (3 ± 1) × 102 (4 ± 1) × 102 (3 ± 1) × 102 (6 ± 2) × 102 (4 ± 1) × 102
Fe (2 ± 2) × 102 (13 ± 7) × 101 (7 ± 3) × 102 (11 ± 6) × 101 (10 ± 4) × 101
Zn (5 ± 2) × 101 (6 ± 2) × 101 (6 ± 2) × 101 (6 ± 1) × 101 (5 ± 2) × 101
Ca/P (5 ± 1) × 10−2 (6 ± 1) × 10−2 (7 ± 3) × 10−2 (9 ± 3) × 10−2 (5 ± 2) × 10−2
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The calcium concentration scan (Figure 3c) clearly shows a limited number of very small calcium-rich spots in the medial layer of the low shear stress region. The size of the hotspots commonly does not exceed the size of one pixel and, hence, their average diameter is 3 μm or less. No phosphorus hotspots could be observed in the medial layer (data not shown).

Figure 4a–d show a distinct calcium hotspot selected at random from a region rich in microcalcifications in the low-shear stress-induced intimal lesion (Figure 2b, area marked with X). Both sulphur and carbon concentrations (Figure 4a and b, respectively) show a decline at the site of the calcium hotspot, indicating that cellular and/or extracellular organic material is replaced by calcium-rich material (Figure 4c). The increase in phosphorus concentration (Figure 4d) merges with the locally increased calcium concentration (Figure 4c).

Figure 4.

Figure 4

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Element concentration distributions, obtained with the proton microprobe, of the area marked with X in Figure 2b, in the thickened, atherosclerotic intima of the low shear stress region. Values at the right hand side of the colour bars refer to concentration (μg/g dry weight of tissue) of sulphur (S; a), carbon (C; b), calcium (Ca; c) and phosphorus (P; d). The dimension of the scan is 29 × 29 μm2; beam size is 0.8 × 0.7 μm2 with an average current 179 pA and a cumulative charge of 11 μC per scan. Pixel size amounts to 0.9 × 0.9 μm2.

Fifteen other calcium hotspots in the thickened intima were randomly selected and analysed with the proton microprobe at a higher magnification. The analysis revealed qualitatively comparable carbon, sulphur and phosphorus concentration distributions (data not shown) as found in the at random chosen calcium micro-precipitation described in the hotspot shown in Figure 4. The combined findings indicate that the intimal microcalcifications are composed, at least, of calcium and phosphorus atoms. The size of these 16 intimal calcium hotspots, investigated in more detail, varies from 6 to 70 μm in diameter.

To address the question whether the calcium hotspots are composed of hydroxyapatite, the main calcium salt of mature bone tissue, calcium/phosphorus mass ratios were assessed in the 16 selected calcium hotspots in the thickened intima. Figure 5a and b show two typical examples of microcalcifications, in which the relation between the calcium and phosphorus concentration is expressed on basis of individual pixels belonging to the distinct microcalcification.

Figure 5.

Figure 5

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Calcium concentration ([Ca]; μg/g dry weight of tissue) plotted vs. phosphorus concentration ([P]; μg/g dry weight of tissue) in individual pixels belonging to two distinct calcium hotspots present in the thickened intima of the low shear stress region of the mouse carotid artery. A pixel was considered to belong to a microcalcification if its concentration exceeded 3 standard deviations from the average calcium concentration measured in the intimal layer remote from calcium hotspots (see Table 1). The drawn line is the linear fit through all pixels composing the individual hotspot. The Ca/P mass ratios, derived from the slopes of the graphs in Figure 5a and b, amount to 1.3 ± 0.1 (R = 0.94) and 4.8 ± 0.2 (R = 0.90), respectively. The errors refer to the uncertainties in the coefficient of the linear fit of the Ca/P plot.

The data indicates that the calcium and phosphorus concentrations inside the individual calcium hotspot show a linear relationship, indicating a chemically homogenous calcium-phosphate crystal. It is of note that the calcium/phosphorus mass ratio in the two examples depicted in Figure 5a and b deviates significantly from that of hydroxyapatite, being 2.16. The Ca/P mass ratio of the hotspots in Figure 5a and b amounted to 1.3 ± 0.1 and 4.8 ± 0.2, respectively. Figure 6 shows the Ca/P mass ratios of all 16 intimal microcalcifications analysed, in ascending order. The values range from about 0.3 to 4.8, which makes it highly unlikely that all calcium deposits are composed solely of hydroxyapatite. It is, however, of note that the median value of the 16 individual Ca/P mass ratios amounted to 2.2.

Figure 6.

Figure 6

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Ca/P mass ratios of 16 individuals, randomly selected calcium hotspots present in the thickened intima of the low shear stress region of mouse carotid artery. Values are represented in ascending order. Closed squares refer to the mean value of all pixels belonging to the distinct microcalcification, vertical lines to the standard error of the mean. These errors refer to the uncertainties of the coefficient of the linear fit of the Ca/P plots.

Next the question was addressed whether the calcium hotspots are surrounded by a region with elevated calcium levels or represent small distinct areas with an abnormally high and steeply increasing calcium concentration. To this end, line scans were made through the thickened intima of the low shear stress region. A typical example of such a mono-dimensional scan hitting several microcalcifications is shown in Figure 7, where the measured area is 512 by 1 pixels.

Figure 7.

Figure 7

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Calcium concentration ([Ca]; μg/g dry weight of tissue) profile plotted vs. position (μm) on a line scan through the thickened intima of the low shear stress region of the mouse carotid artery.

In general, the calcium concentrations in areas surrounding microcalcifications were in the order of (4 ± 1) 102 μg/g dry weight of tissue (Table 1). This value was not significantly different from the calcium concentration assessed in the vessel wall of the region with high shear stress (varying from 300 to 400 μg/g dry weight of tissue) and previously found in healthy cardiac tissue as measured with a similar proton microprobe technique (Verhoef et al. 1996). Detailed analysis showed that the calcium concentration steeply increased in close vicinity of a calcium hotspot. In one occasion (hotspot around position 340 μm; Figure 7), the calcium concentration increased even 150-fold within 2–4 μm.

Trace elements iron and zinc

The present proton microprobe analysis of vascular tissue allows for analysing the tissue concentration of trace elements such as iron and zinc. First, we determined the tissue concentration of these elements in regions remote from depositions of calcium-rich material (Table 1). Furthermore, it is of note that occasionally spots with increased concentrations of iron and zinc were observed in the thickened intima of the low shear stress region. The co-localization, if any, of these two elements with calcium was investigated on detailed scans made of 16 microcalcifications and their immediate surroundings. A typical example of such scans is shown in Figure 8a–c.

Figure 8.

Figure 8

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Element concentration distributions, obtained with the proton microprobe, of one at random selected calcium hotspot in area X in Figure 2b of the intimal atherosclerotic lesion. Values at the right hand side of the colour bars refer to concentration of calcium (Ca, a), iron (Fe, b) and zinc (Zn, c), expressed as μg/g dry weight of tissue. The dimension of the scan is 29 × 29 μm2; beam size is 0.8 × 0.7 μm2 with an average current of 179 pA and a cumulative charge of 11 μC per scan. Pixel size amounts to 0.9 × 0.9 μm2. The concentration of the pixels below the detection level is set at 0 μg/g and these pixels are coloured white (e.g. b and c). The detection level for Fe and Zn was found to be 24 ± 12 μg/g and 16 ± 15 μg/g, respectively.

In this example, enhanced concentrations of iron and zinc show an excellent co-localization with the calcium hotspot. Figure 9, representing a typical line-scan through the thickened intimal layer displaying a big and small calcium hotspot, clearly shows the correspondence in accumulation pattern between iron and zinc.

Figure 9.

Figure 9

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Concentration (μg/g dry weight of tissue) profiles of calcium, iron and zinc plotted vs. position on a line scan through the area marked with X (Figure 2b) in the thickened intima of the low shear stress region of mouse carotid artery. The two sections in pale grey refer to distinct calcium hotspots.

Detailed analysis of the 16 individual calcium microcalcifications showed that in the majority of cases increased concentrations of iron and zinc coincided with increased concentrations of calcium, i.e. 75% and 88%, respectively, of the microcalcifications investigated.

Discussion

This study shows that microcalcifications are present in the atherosclerotic lesion of the APOE−/− murine carotid artery, in a region subjected to low shear stress. The calcium hotspots were more abundant and bigger in size in the intimal than medial layer of the vascular wall. Our findings are in line with the notion that calcium precipitation has its origin in small and well defined loci in the affected tissue. This conclusion is based on observations in line scans made with the proton microprobe through the thickened intima. The calcium micro-deposits appear to be localized in areas with calcium concentrations not different from non-atherosclerotic biological material. Occasionally, the calcium concentration of the microcalcification steeply increased, at least 150 times that of the surrounding area within a couple of micrometres.

Mass ratio of calcium and phosphorus in microcalcifications

Detailed information on the mass ratio of calcium and phosphor in the microcalcifications might shed light on the mechanisms underlying the precipitation of calcium salts in the atherosclerotic lesion. Studies of Schmid et al. (1980) on big lumps of calcified material in human aortas with advanced atherosclerosis indicated that the Ca/P mass ratio resembled that of pure hydroxyapatite, i.e. 2.16. Later studies on human coronary arteries (Fitzpatrick et al. 1994) supported their observation. The corollary of these findings was that the mechanism underlying calcification of arterial walls is closely linked to the process of bone formation (Donley & Fitzpatrick 1998; Doherty et al. 2002; Fitzpatrick et al. 2003). Contrasting data, however, were published by McCormick et al. (2005) and Becker et al. (2004), reporting values below 2.16, suggesting the presence of calcium-deficient hydroxyapatite. The present detailed analysis of 16 randomly selected calcium hotspots in the thickened intima revealed that the Ca/P mass ratio varied between 0.3 and 4.8. The broad range of ratios suggests that, at the micrometre scale, calcium hotspots are composed of amorphous calcium phosphate crystals rather than pure hydroxyapatite, although it cannot be excluded that occasionally dicalcium phosphate dehydrate, octacalcium phosphate and Mg-substituted tricalcium phosphate (Ca/P mass ratios of 1.29, 1.72 and 1.94, respectively) crystals or a combination thereof are also present. Since the Ca/P mass ratio of amorphous calcium phosphate crystals reportedly varies between 1.55 and 3.23 (LeGeros 2001; Tomazic 2001), values above 3.23 suggest the presence of other anions such as urate or carbonate. It should be emphasized that the median value of the Ca/P mass ratios in the 16 individual microcalcifications was found to be 2.2. This value is indeed very close to that of pure hydroxyapatite, i.e. 2.16. The outcome of this calculation might explain why investigators analysing calcium and phosphorus levels in lumps of calcified material, using techniques with very limited resolution, are reporting values corresponding with the Ca/P mass ratio of hydroxyapatite. Here we show that on the pixel base the chemical composition of the individual calcium hotspots appears to be very homogenous, strongly suggesting that each single microcalcification is composed of only one type of calcium crystal. This conclusion is in line with the outcome of an earlier study on microcalcifications in atherosclerotic human coronary arteries (Roijers et al. 2008). The broad range of Ca/P mass ratios in individual calcium hotspots may challenge the notion that calcium deposition in atherosclerotic lesions exactly mimics the formation of bone tissue. Other mechanisms are most likely also involved, the nature of which should be clarified in future studies. It is of note that additional analytical techniques such as electron diffraction are necessary to confirm the presence or absence of hydroxyapatite in the calcium hotspots present in the atherosclerotic lesions.

Co-localization of trace elements iron and zinc with micro-calcifications

Here it was shown that in the majority of cases deposition of calcium coincides with locally increased concentrations of iron and zinc. Increased concentrations in iron have been observed earlier in advanced atherosclerotic lesions (Lee et al. 1998; Makjanic et al. 1998; Stadler et al. 2004; Langheinrich et al. 2007). Pioneering studies of Pallon et al. (1995) revealed that iron accumulations in atherosclerotic lesions of the human coronary artery were occasionally surrounded by calcium-rich material. Iron is supposed to play a detrimental role in the atherosclerotic process (Minqin et al. 2003; Stadler et al. 2004). Locally accumulated iron is thought to provoke, among others, peroxidation of lipids, either internalized by macrophages or deposited in the interstitial compartment (Gaut & Heinecke 2001; Stocker & Keaney 2004).

In the past, both decreased (Makjanic et al. 1998; Minqin et al. 2003) and enhanced (Pallon et al. 1995) concentrations of zinc have been observed in atherosclerotic lesions. The role of zinc, if any, in the atherosclerotic process is incompletely understood. Since zinc is a cofactor of the enzyme superoxide dismutase, this element could prevent excessive lipid peroxidation by facilitating oxygen free radical scavenging (Stocker & Keaney 2004).

Limitations of the study and future directions

The present, explorative study was performed on two sets of tissue sections, one from the atherosclerotic low shear stress and one from the non-atherosclerotic high shear stress region, of a carotid artery obtained in one randomly selected APOE−/− mouse, representing a well-established murine model of atherosclerosis. Only one randomly selected animal was used for the micro-probe analysis because one of the main aims of the study was to provide a proof of principle that the present analytical technique, characterized by a relatively high resolution, is useful to determine the alterations, if any, in the element concentration of diseased mouse vascular tissue.

Despite the limited number of biological samples it is felt that the present findings provide relevant information on the presence of microcalcifications, and the co-localization of trace elements, such as iron and zinc, in the thickened intima of the atherosclerotic lesion. The present demonstration of micro-deposits of calcium and trace elements with the proton microprobe technique offers the opportunity to use this murine model to investigate in more detail the time course of the precipitation of calcium salts and associated trace elements after the induction of a low-shear region in the carotid artery. Moreover, the size of the proton beam allows for investigating the site of calcium precipitation, i.e. cellular or extracellular. The nature of the vascular cells involved can be identified by combining the proton microprobe technique with immuno-histochemical analysis of adjacent tissue sections. These findings may help to deepen our insight in the pathophysiological significance of microcalcifications in the sequel of events resulting in advanced atherosclerotic lesions and the mechanisms underlying the process of calcium phosphate deposition in the affected artery wall.

Acknowledgments

The authors are indebted to Caroline Cheng and Dennie Tempel (Erasmus University Rotterdam) for providing biological material. Financial support was received from ‘Stichting voor Fundamenteel Onderzoek der Materie’ (FOM), project OOPMT13, for executing the investigation.

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