Matrix vesicles in the fibrous cap of atherosclerotic plaque: possible contribution to plaque rupture

Abstract

Plaque rupture is the most common type of plaque complication and leads to acute ischaemic events such as myocardial infarction and stroke. Calcification has been suggested as a possible indicator of plaque instability. Although the role of matrix vesicles in the initial stages of arterial calcification has been recognized, no studies have yet been carried out to examine a possible role of matrix vesicles in plaque destabilization. Tissue specimens selected for the present study represented carotid specimens obtained from patients undergoing carotid endarterectomy. Serial frozen cross-sections of the tissue specimens were cut and mounted on glass slides. The thickness of the fibrous cap (FCT) in each advanced atherosclerotic lesion, containing a well developed lipid/necrotic core, was measured at its narrowest sites in sets of serial sections. According to established criteria, atherosclerotic plaque specimens were histologically subdivided into two groups: vulnerable plaques with thin fibrous caps (FCT <100 microm) and presumably stable plaques, in which fibrous caps were thicker than 100 microm. Twenty-four carotid plaques (12 vulnerable and 12 presumably stable plaques) were collected for the present analysis of matrix vesicles in fibrous caps. In order to provide a sufficient number of representative areas from each plaque, laser capture microdissection (LCM) was carried out. The quantification of matrix vesicles in ultrathin sections of vulnerable and stable plaques revealed that the numbers of matrix vesicles were significantly higher in fibrous caps of vulnerable plaques than those in stable plaques (8.908+0.544 versus 6.208+0.467 matrix vesicles per 1.92 microm2 standard area; P= 0.0002). Electron microscopy combined with X-ray elemental microanalysis showed that some matrix vesicles in atherosclerotic plaques were undergoing calcification and were characterized by a high content of calcium and phosphorus. The percentage of calcified matrix vesicles/microcalcifications was significantly higher in fibrous caps in vulnerable plaques compared with that in stable plaques (6.705+/-0.436 versus 5.322+/-0494; P= 0.0474). The findings reinforce a view that the texture of the extracellular matrix in the thinning fibrous cap of atherosclerotic plaque is altered and this might contribute to plaque destabilization.

Fig 1

Histologically identifiable large and huge calcified deposits visualized by means of routine histology (A; Haematoxylin & eosin staining) and histo-chemistry using Alizarin red S (B–E) and von-Kossa techniques (F, G). (D) is a detail of (C). In (A, B, F and G), L, arterial lumen; FC, fibrous cap; NC, necrotic core. In (A), star indicates an area where a huge calcified deposit has been formed; this huge calcified deposit was lost during tissue sectioning. Scale bars: 400 μm (A), 250 μm (B, F), 50 μm (C, E), 150 μm (G).

Fig 2

Typical appearance of matrix vesicles in the fibrous cap of atherosclerotic plaque (A-H). (A-B): Matrix vesicles surrounded by two electron-dense layers (lamellae) and filled with granular (A) or homogenous material (B) of medium electron density. (D-H): Matrix vesicles covered by multiple lamellae. (D) is a detail of (C). (F) is a detail of (E). (H) is a detail of (G). Transmission electron microscopy (TEM). Scale bars: 50 nm (A, B, C, EandG).

Fig 3

Examples of images of matrix vesicles located in fibrous caps in vulnerable (A) and stable plaques (B), which were used for the quantitative analysis (see Methods section): TEM. Scale bars: 200 nm (A-B). (C): Image showing an example of laser capture microdissection of tissue samples from a fibrous cap. Scale bar: 25 μm (D): Numbers/relative densities of matrix vesicles in fibrous caps in vulnerable (A) and stable plaques (B).

Fig 4

Typical appearance of matrix vesicles undergoing calcification (A-G). In (A), the large arrow shows a calcifying matrix vesicle that is characterized by a very high electron density while the small arrow shows a non-calcified matrix vesicle that displays a medium electron density. In (B), the arrow shows the zone of a calcifying matrix vesicle where the association of calcified deposits with the multilamellar structure is evident. Note that calcification occurs inside the matrix vesicle as well as along the vesicle contour. (C) is a detail of (B). In (D-F), spicules of calcium salt crystals are distributed along the contour of a matrix vesicle where they are associated with lamellar structures (shown by arrows in Fig. E and F). (E) and (F) are details of (D). In (G), spicules of calcium salt crystals are distributed irregularly throughout the matrix vesicle. TEM. Scale bars: 100 nm (A, B, D, G). (H): X-ray elemental microanalysis showing the presence of calcium and phosphorus in a calcifying matrix vesicle.