A mechanistic analysis of the role of microcalcifications in atherosclerotic plaque stability: potential implications for plaque rupture

Abstract

The role of microcalcifications (μCalcs) in the biomechanics of vulnerable plaque rupture is examined. Our laboratory previously proposed (Ref. 44), using a very limited tissue sample, that μCalcs embedded in the fibrous cap proper could significantly increase cap instability. This study has been greatly expanded. Ninety-two human coronary arteries containing 62 fibroatheroma were examined using high-resolution microcomputed tomography at 6.7-μm resolution and undecalcified histology with special emphasis on calcified particles <50 μm in diameter. Our results reveal the presence of thousands of μCalcs, the vast majority in lipid pools where they are not dangerous. However, 81 μCalcs were also observed in the fibrous caps of nine of the fibroatheroma. All 81 of these μCalcs were analyzed using three-dimensional finite-element analysis, and the results were used to develop important new clinical criteria for cap stability. These criteria include variation of the Young's modulus of the μCalc and surrounding tissue, μCalc size, and clustering. We found that local tissue stress could be increased fivefold when μCalcs were closely spaced, and the peak circumferential stress in the thinnest nonruptured cap (66 μm) if no μCalcs were present was only 107 kPa, far less than the proposed minimum rupture threshold of 300 kPa. These results and histology suggest that there are numerous μCalcs < 15 μm in the caps, not visible at 6.7-μm resolution, and that our failure to find any nonruptured caps between 30 and 66 μm is a strong indication that many of these caps contained μCalcs.

Fig. 1.

High-resolution microcomputed tomography (HR-μCT) image compared with undecalcified histological 5-μm-thick sections. A: 6.7-μm-resolution HR-μCT image showing bright calcifications of different sizes (scale bar is 500 μm). B: corresponding section stained with von Kossa showing phosphate deposits (×1, scale 500 μm). C: consecutive section stained with Alizarin Red S (×1, scale 500 μm) shows calcified areas. D: detailed area in B, showing microcalcifications (μCalcs) of 1–10-μm size in a cap (scale bar is 20 μm).

Fig. 2.

A: mean number of calcifications in human coronary arteries (n = 92) classified by size. μCalcs < 50 μm are shown in shaded bar. B: percentage of the total number of calcifications in each size group. Comparison of total number of calcifications within these three groups was found significantly different (*P < 0.05). LAD, left anterior descending artery; LCX, left circumflex artery; RCA, right coronary artery. Values are means ± SD.

Fig. 3.

A: HR-μCT image of an atheroma showing calcifications of different sizes. B: applying a double threshold 450–850 mg/cm³, it is possible to separate μCalcs (shown in red) from those of larger size (bright green), indicating the differences in the degree of mineralization. Scale bar is 500 μm. C: tissue mineral density (TMD) of μCalcs vs. large calcifications. The degree of mineralization is significantly higher in large calcifications (*P < 0.004). D: calcified volume fraction (CVF). The mean CVF is highest in the LAD (*P < 0.01). Values are means ± SD. ns, Nonsignificant.

Fig. 4.

Spatial distribution of μCalcs along the longitudinal axis of the artery in the LCX (top), LAD (middle), and RCA (bottom), measured from the ostium to the centroid of the μCalc. Values are means ± SD.

Fig. 5.

A: three-dimensional reconstruction of an artery segment with a fibroatheroma, showing calcifications of various sizes, and the corresponding two-dimensional inverse gray-scale HR-μCT image showing μCalcs in the fibrous cap proper (dark spots). Scale bar is 500 μm. B: an approximately spherical μCalc in the cap corresponding to the highlighted area in A. Regions of high stress concentration appear in the cap at the poles of the μCalc, creating a 2.1-fold increase in the local stress in the above sample.

Fig. 6.

Stress distribution calculated in a cap, where four μCalcs are visible at the same level, showing how calcifications in close proximity introduce a high-stress concentration of about five times the circumferential stress of the background, while relatively isolated μCalcs increase the PCS a factor of two.

Fig. 7.

Effect on PCS of a 25-μm μCalc embedded in a fibrous cap of 120 μm thickness for twofold changes in elastic moduli (intimal stiffness). Changes in the intimal stiffness increase peak circumferential stress ∼30%, whereas the presence of a μCalc increases peak circumferential stress >80%.

Fig. 8.

HR-μCT image of a culprit ruptured fibrous cap showing calcifications in black and soft tissue in light gray. The arrowhead points to a μCalc present at the rupture site. Interfacial debonding at the interface of the μCalc would have caused the atheroma to rupture (scale bar is 500 μm).